Phospholipid compositions

ABSTRACT

Compositions involving a modified egg yolk extract for use as an effective anti-cancer agent are described. The modified egg yolk extract involves specific fractions of phosphatidylcholines and sphingomyelins modified and produced from a chemical synthesis applied to the extract that produce a beneficial effect on the inhibition of cancerous cell growth. Methods of administering these compositions are also described.

This application is a continuation of, and claims priority under 35U.S.C. §120 to, U.S. patent application Ser. No. 15/637,126 filed Jun.29, 2017, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application Ser. Nos. 62/356,189 and 62/356,197, eachfiled Jun. 29, 2016. The entire contents of each of the foregoingapplications are hereby incorporated by reference herein.

FIELD OF INVENTION

Pharmaceutical compositions comprising bioactive lipids and methods oftreating mammals with bioactive lipids are disclosed. The bioactivelipids generally include phosphatidylcholine (PC) and/or sphingomyelin(SPH) compounds and are believed to be useful in the treatment ofproliferative diseases including cancer.

BACKGROUND

Some phospholipids from natural sources are known to produce beneficialeffects on various conditions in mammals. For example, phospholipidsderived from hen egg yolk have been studied as potentially havingtherapeutic benefits. Natural hen egg yolk extracts are rich in avariety of bioactive phospholipids (BAP). When enriched to 30% with theN-acyl ether-phosphatidylethanolamine (NAEPE)1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamine,hen egg yolk extracts have been reported to have anti-cancer properties,including significantly interfering with tumor progression in in vivochick models. See, Karafiát V., et al., Folia Biologica (Praha) 60,220-227 (2014). This is consistent with earlier reports that1-O-octadecyl/hexadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamineisolated from ischemic chicken embryos and/or made semisyntheticallyrestricts growth of subcutaneous transplanted sarcomas in mice. See,Kara, J. et al., Neoplasma 33, 187-205 (1986) and Kara J. et al.,Neoplasma 40 213-217 (1993).

It is generally believed that the alkyl-ether moiety in NAEPE isnecessary for cytotoxic activity because ether-linked lipids are notcatabolized in tumors and can accumulate and interfere with vitalpathways of the cell. See, Kara, J. et al, Neoplasma 33, 187-205 (1986),Berdel, W. et al., Cancer Res. 43, 541 (1983), and Modolell, M., et al.,Cancer Res., 39 4681 (1979).

Unlike classical DNA-targeted cytotoxic agents, alkylphospholipids orBAP may target cellular and intracellular membranes. See Kuerschner, D.,et al., PLOS One 7, e31342 (2012). When administered in therapeuticdoses, alkylphospholipids may inhibit phosphatidylcholine biosynthesis,interfere with lipid transduction pathways, block the endoplasmicreticular transport, and interfere with the membrane lipid raftfunction. See, e.g., Blitterswijk. W., et al., Biochim. Biophys. Acta.1831, 663-674 (2013). Lipid rafts are specialized plasma membranemicrodomains having concentrations of cholesterol and sphingomyelinswhich spatially organize signaling pathways and regulate cellproliferation and apoptosis. See, e.g., van der Luit, A., et al., Mol.Cancer Ther. 6, 2337-2345 (2007). Lipid rafts are more abundant incancer cells relative to normal cells and have been proposed to serve asentry points for cytotoxic agents into the cells. See, e.g., Li, Y., etal., Am. J. Pathol. 168, 1107-1118 (2006).

In addition to potential anti-neoplastic properties, it has also beenreported that ischemic chick embryonic tissue extract enriched to 30%1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)ethanolamineshows significant anti-inflammatory and immunomodulatory effects. See,Vicenova, M. et al., Complementary and Alternative Medicine 14 339(2014). In in vivo studies of bacterially induced acute pneumonia it wasdemonstrated that BAP mixtures enriched with NAEPE had a positive effecton disease progression by lowering levels of IL-1β, IL-8 in sera andlowering white blood count, as well as reducing lung parenchyma. Invitro studies on the transcriptional activity of proinflammatorycytokine genes related to the activation of intracellular signalingpathways associated with inflammation showed the ability of certain BAPmixtures to influence the immune response of macrophages. Id. Moreover,the NAEPE enriched egg yolk extracts have activities which inhibit thephosphorylation of protein kinase C epsilon. Id.

It is an object of the present invention to identify biologically activelipids, including phosphatidylcholine (PC) and/or sphingomyelin (SPH)and/or lysophosphatidylcholine (LPC) compounds, and mixtures thereof,possessing therapeutic properties, including antiproliferative orantineoplastic properties. Moreover, it is an object of the invention toprovide compositions and methods of using these compositions to treatproliferative diseases, including cancer.

SUMMARY OF INVENTION

Bioactive phospholipids (BAP) can be extracted from hen egg yolk (e.g.,ischemic chick embryos) with alcohol and can be purified using acetoneprecipitation, as disclosed in Gladkowski, W, et al., J. Am. Oil Chem.89 179-182 (2012), hereby incorporated by reference in its entirety.Such an egg yolk extract (designated herein as “BAP(−)”) contains a widerange of phospholipids but, as demonstrated herein, does not showsignificant effects in in vitro and in vivo studies of cancer. It isknown to treat BAP(−) mixtures with palmitoyl chloride to produce amaterial known as BAP(+). It has heretofore been believed thatpalmitoylization of the BAP(−) extract (producing BAP(+)) is required toyield therapeutic activity of the BAP mixture due to the creation ofNAEPE's (or “PNAE”) from the corresponding endogenous ethanolamines. Asdiscussed previously, NAEPE's were thought to be the active constituentin BAP(+). See, e.g., Karafiat V., et al., Folia Biologica (Praha) 60,220-227 (2014) and Kara, J. et. al, Neoplasma 33, 187-205 (1986).

However, it has surprisingly been discovered that certain lipidcomponents of chemically treated hen egg yolk provide anti-cancereffects, even in the absence or substantial absence (e.g., less than 5%(w/w), less than 1% (w/w), less than 0.1% (w/w), less than 0.01% (w/w),etc.) of the known active NAEPE's. Accordingly, the invention providesnovel mixtures of lipids and pharmaceutical compositions thereof whichare contemplated to be useful in the treatment of various diseases andconditions. In some aspects, the invention provides compositionscomprising egg yolk extracts (e.g., hen egg yolk extracts, typicallyfrom Gallus gallus domesticus) enriched with one or more lipidcomponents, such as phosphatidylcholine (PC) and/or sphingomyelin (SPH)and/or lysophosphatidylcholine (LPC) compounds. The invention is notlimited to hen egg yolk extracts, but embraces extracts from any animalsource or synthetic mixtures of lipids, having the same or substantiallysimilar constituents. By “substantially similar” constituents is meantthat at least the most abundant PC and/or SPH components are within 25%(w/w), preferably 15% and ideally 10% of the abundance in the hen eggyolk extracts defined herein. In some implementations, one or morelipids according to the invention are inhibitors of kinases, includingprotein kinases, notably tyrosine kinases, such as TTK. Thesecompositions may be used to treat, for example, inflammation orproliferative diseases such as cancer.

Methods of treating proliferative diseases are also provided. In oneaspect, a method for the treatment of a mammal (e.g., a human) sufferingfrom cancer are provided comprising administering therapeuticallyeffective amounts of any of the lipid mixtures described herein, orpharmaceutical compositions thereof. In particular, the lipid mixturesare derived from egg yolk extracts (e.g., hen egg yolk extracts, etc.)enriched in phosphatidylcholine and/or sphingomyelin compounds and/orlysophosphatidylcholine compounds or synthetically derived mixtures thatare substantially similar thereto. In various implementations, thecompositions are administered (e.g., orally) for the treatment of acancer, including, without limitation, bladder, blood (e.g. lymphoma,Jurkat cancer cell line, etc.) brain (e.g. T98G cancer cell line, etc.),breast (e.g. 231 cancer cell line, etc.), cervical (e.g. HeLa cancercell line, etc.), colorectal (e.g. HCT116 cancer cell line, etc.),esophageal, kidney, liver (e.g. HepG2 cancer cell line, etc.), lung(e.g. H358 cancer cell line, A549 cancer cell line, etc.), ovarian (e.g.SK-OV-3 cancer cell line, etc.), pancreatic (e.g. Pancl cancer cellline, Capan-2 cancer cell line, etc.), skin (e.g. M14 cancer cell line,etc.), prostate (e.g. DU-145 cancer cell line, etc.), thyroid, oruterine cancers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an isolation of specific set of phospholipids derivedfrom an egg extract. The initial egg yolk mixture comprises O-alkylPE's, SPH's, PC's and other constituents. It will be understood that “R”groups on any compound class in FIG. 1 may be any aliphatic radical, andmore typically, the monovalent hydrocarbon radical of a naturallyoccurring fatty acid. In the derivation illustrated, phospholipids areextracted from egg yolk using butanol (step 1) and further purified byadding the butanol extract to chilled acetone and collecting the lipidswhich precipitate from the mixture (step 2). These precipitated lipidsare then reacted with CDI and an acid (for example, using an ethylacetate:triethanolamine solvent comprising an amount of abouttriethanolamine for complete solvation of the precipitated lipids). Suchreaction may produce the ten lipid classes shown. Medium pressurechromatography (step 4) is then used to further isolate only specificlipid classes (e.g. SPH, PC, LPC) and the final product may then befurther purified using water/chloroform extraction to remove residualdegradation products produced during chromatographic separation (step4).

FIG. 2A shows TLC plates for a thin layer chromatographic separation ofBAP(+), BAP(−) into various fractions from the left side of the plate tothe solvent front on the right. Stains from fractions F1-F5 of BAP (+)and the corresponding fraction F5 of BAP(−) (“F5(−)”) may be seen. Ascan be seen, the acylation BAP(−) to produce BAP(+) converts the PEcomponent of BAP(−) into an NAEPE component present in BAP(+). Alsoshown are SPH, LPC and PC controls illustrating where those phospholipidclasses elute. As can be seen, fraction F5 comprises a sphingomyelincomponent and a phosphatidylcholine component. FIG. 2B shows a similarchromatographic separation of MP1000 (top trace) with LPC, SPH andBAP(+) controls. As can be seen, MP1000 is composed of the F5 fractionbut an LPC component is substantially absent. FIG. 2C shows thechromatographic comparison of MP1000, to BAP(+), and BAP(−) controls.

FIG. 3A shows an HPLC chromatogram of fraction MP1000 and demonstratesthe isolation and separation of further sub-fractions P1, P2, P3, P4,P5, P6, and P7. Additionally, retention times and area under curve foreach sub-fraction are shown. FIG. 3B shows the HPLC chromatogram forFraction F5 with sub Fractions P1-P7 identified.

FIG. 4 illustrates a chromatogram of MP1000 separation using normalphase HPLC protocol which separates the lipid species. The peak elutingat 6.0 minutes corresponds to the phosphatidylcholine component and thepeak eluting at 12.2 minutes corresponds to the sphingomyelin component.The peak eluting at 2.2 minutes corresponds to the solvent front in thischromatographic extraction and does not correspond to any part ofMP1000.

FIG. 5 illustrates a gas chromatogram of fatty acid methyl esteranalysis of MP1000 and MP1000(−). The RT for several prominent methylesters are given. The MP1000(−) is offset from the zero line forcomparison of the two traces.

FIGS. 6, 7 and 8 illustrate in vivo mouse model tumor growthmeasurements of various cancer cell lines as a result of administrationof various BAP mixtures.

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein;however, it is to be understood that the disclosed embodiments aremerely illustrative of the invention that may be embodied in variousforms. In addition, each of the examples given in connection with thevarious embodiments of the invention is intended to be illustrative, andnot restrictive.

All terms used herein are intended to have their ordinary meaning unlessotherwise provided. As used herein, the term “consisting essentially of”is intended to limit the invention to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristics of the claimed invention, as understood from a readingof this specification.

Whenever a term is identified by reference to a range, the range will beunderstood to explicitly disclose the endpoints and every element orvalue within the range thereof. The exact values of all half integralnumeric values will also be understood to be specifically disclosed inany range and subsets of the original range. For example, a range offrom about 0.1% to about 3% specifically discloses a percentage of 0.1%,0.5%, 1%, 1.5%, 2%, 2.5%, and 3%, and any subranges formed by thoseintermediate values.

Where two or more substituents are referred to as being “independentlyselected from” a listing of alternatives, it is meant that eachsubstituent can be any element of that group, independent of theidentity of other substituents.

Unless otherwise stated, the phrase “substantially free” refers to anamount of a component that is sufficiently low such that the componentcontributes no significant properties (e.g., bioactivity, etc.) to thebulk, and, in any event will be less than about 5.0% by weight or lessthan about 4.0% or less than about 3.0% by weight or less than about2.0% by weight or less than about 1.0% by weight or less than about 0.5%by weight or less than about 0.25% by weight or less than about 0.1% byweight, based on the total weight of the composition or based on theweight of a given component depending on the context.

As used herein, “% by weight” or “% wt.” or “w/w” refers to the weightpercent of a component in relation to the total weight of thecomposition unless otherwise stated. Every reference to percentage or %herein is given on a % by weight basis, unless stated otherwise. It willbe understood that the sum of all weight % of individual componentswithin a composition or within indicated component will not exceed 100%.

As used herein, the term “about” modifying a quantity refers tovariation in the numerical quantity that can occur, for example, throughtypical measuring and liquid handling procedures used for makingconcentrates or use solutions in the real world; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of the ingredients employed to make the compositionsor carry out the methods; and the like, as understood by persons ofordinary skill in the art. Whether or not modified by the term “about,”the claims include equivalents to the quantities.

A “patient in need thereof,” as used herein, refers to a humanindividual, male or female, who would benefit from administration oftherapeutically effective doses of the lipid compositions. As describedherein, in some embodiments, an individual in need thereof is sufferingfrom a proliferative disorder such as cancer. In some embodiments, anindividual in need thereof has been diagnosed by a medical doctor with aproliferative disorder requiring treatment. A patient in need or anindividual in need are used interchangeably herein.

As used herein, the phrase “pharmaceutically acceptable” generally safefor ingestion or contact with biologic tissues at the levels employed.Pharmaceutically acceptable is used interchangeably with physiologicallycompatible. It will be understood that the pharmaceutical compositionsof the invention include nutraceutical compositions (e.g., dietarysupplements) unless otherwise specified.

The phrase “therapeutically effective amount,” as used herein, means anamount necessary to provide the indicated therapeutic benefit. Forexample, a therapeutically effective amount may be from about 1 mg toabout 10 g administered once (q.d.) or twice (b.i.d.) daily.

It will be understood that the description of compounds herein islimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding with regard to valences, etc., andto give compounds which are not inherently unstable. For example, anycarbon atom will be bonded to two, three, or four other atoms,consistent with the four valence electrons of carbon.

Any of the compounds of the present disclosure may be in the form ofpharmaceutically acceptable salts. “Pharmaceutically acceptable salts,”as used herein, denotes salts that are physiologically compatible, asdefined herein, and that possess the desired pharmacological activity ofthe parent compound. Such salts include: acid addition salts formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like; or formed with organicacids such as acetic acid, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,hydroxynaphtoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleicacid, malic acid, malonic acid, mandelic acid, methanesulfonic acid,muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylicacid, succinic acid, tartaric acid, p-toluenesulfonic acid,trimethylacetic acid, and the like; or salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic or inorganic base. Acceptable organic basesinclude diethanolamine, ethanolamine, N-methylglucamine,triethanolamine, tromethamine, and the like. Acceptable inorganic basesinclude aluminum hydroxide, calcium hydroxide, potassium hydroxide,sodium carbonate, and sodium hydroxide.

As used herein, an “enriched” extract is meant that the relativeabundance of a given phospholipid in relation to any other phospholipidsin the composition is increased in the extract compared to the relativeabundance of the same phospholipid to the same other phospholipids inthe parent composition from which the extract is derived.

As used herein, “hen egg yolk extract” refers to the yolk extract of anyavian species. In some embodiments, the hen egg is from a species fromthe genus Gallus. In some embodiments, the hen egg is from the speciesGenus gallus. In other embodiments, the hen egg is from the speciesGallus gallus domesticus.

A large portion of the lipids found in hen egg yolk are phospholipidscomprising a choline head group which include alkylphospholipids andphosphatidylcholine (PC) compounds. These choline comprisingphospholipids generally have the structure of formula I(a):

where R₁ and R₂ can be independently any monovalent C₁-C₃₀ radical, butare typically independently groups R or R—C(O)— where R is an aliphatichydrocarbon chain, and more typically corresponding to naturallyoccurring fatty acids (or the acyl portion thereof). The acyl groups maycorrespond to the acyl portion of naturally occurring fatty acids suchas ω-3 and ω-6 fatty acids. As used herein, a reference to a lipid thatcomprises a fatty acid will be understood to refer to the acyl group ofthe indicated fatty acid (i.e., where the lipid is esterified with thefatty acid), unless otherwise specified. For example, aphosphatidylcholine of formula I(a) comprising a docosahexaenoic fattyacid (e.g., (C22:6, all cis-4,7,10,13,16,19), etc.) has adocosahexaenoyl acyl group at position R₁ and/or R₂.

Some naturally occurring fatty acids can provide beneficial effects tocells, but are not naturally occurring in many mammalian bodies. Fattyacids (of the form R—COOH) typically have a long aliphatic chain (R),which is normally saturated, unsaturated, or poly-unsaturated, connectedto a carboxylic acid (—COOH) head group. The corresponding acyl groupwill therefore have the form R—(CO)—. Most naturally occurring fattyacids have an unbranched hydrocarbon chain of an even number of carbonatoms (typically from 4 to 28). A fatty acid (or acyl radical thereof)may be denoted by its lipid number which indicates both the length ofhydrocarbon chain of a fatty acid and the number of double bonds in thatchain. In some embodiments, any double bond may be in the cis or trans(or E or Z) configuration. In some embodiments, all double bonds withina given fatty acid (or acyl fragment thereof) will be in the cisconfiguration. For example, a (C22:5) fatty acid has an unbranchedhydrocarbon chain with a length of 22 carbon atoms and 5 double bondsand the corresponding C22 acyl radical has the structure RC(O)—, where Ris a hydrocarbon chain with a length of 21 carbons and having 5 doublebonds. ω-n numbers on fatty acids indicate the carbon distance (n) fromthe terminal methyl carbon (w) on the fatty acid where the first doublebond is located. An unsaturated fatty acid may be designated by thelocation of each double bond and the isomeric configuration of that bond(e.g., cis or trans). The location of the double bond may also bedesignated, for example, through the carbon number from which the doublebond is located and extending toward the terminal methyl carbon. Unlessotherwise specified, the carbon number is used to designated the doublebond position. For example, a (C22:5, all cis-7,10,13,16,19) fatty acidacyl group has the structure:

Unless otherwise indicated, successive double bonds are assumed to beseparated by a single methylene (—CH₂—) unit. Table 1 shows a commonname, systematic name, and structure of some fatty acids (andcorresponding acyl portions thereof) that may be suitable R₁ and/or R₂substituents.

TABLE 1 Common Name (Lipid Number) Systematic Name Structure Crotonicacid (C4:1) (E)-2-Butenoic acid

Myristic acid (C14:0) Tetradecanoic acid

Myristoleic acid (C14:1) (Z)-9-Tetradecenoic acid

Palmitic acid (C16:0) (Z)-9-Hexadecenoic acid

Palmitoleic acid (C16:1) (Z)-9-Hexadecenoic acid

Sapienic acid (C16:1) (Z)-6-Hexadecenoic acid

Margaric Acid (C17:0) Heptadecanoic acid

Stearic acid (C18:0) Octadecanoic acid

Elaidic acid (C18:1) (E)-9-Octadecenoic acid

Oleic Acid (C18:1) (Z)-9-Octadecenoic acid

cis-Vaccenic acid (C18:1) (Z)-11-Octadecenoic acid

Vaccenic acid (C18:1) (E)-11-Octadecenoic acid

Linoleic acid (C18:2) (9Z,12Z)-9,12- Octadecadienoic acid

α-Linolenic acid (C18:3) (9Z,12Z,15Z)-9,12,15- Octadecatrienoic acid

Γ-Linolenic acid (C18:3) (6Z,9Z,12Z)-6,9,12- Octadecatrienoic acid

Pinolenic acid (C18:3) (5Z,9Z,12Z)-5,9,12- Octadecatrienoic acid

α-Eleostearic acid (C18:3) (9Z,11E,13E)-9,11,13- Octadecatrienoic acid

β-Eleostearic acid (C18:3) (9E,11E,13E)-9,11,13- Octadecatrienoic acid

Stearidonic acid (C18:4) (6Z,9Z,12Z,15Z)- 6,9,12,15- Octadecatetraenoicacid

Bosseopenta- enonic acid (C18:5) (5Z,8Z,10E,12E,14Z)- 5,8,10,12,14-Eicosapentaenoic acid

Nonadecylic acid (C19:0) Nonadecanoic acid

Arachidic acid (C20:0) Eicosanoic acid

Gadoleic acid (C20:1) (9Z)-9-Eicosenoic acid

Gondoic acid (C20:1) (Z)-11-Eicosenoic acid

Eicosadienoic acid (C20:2) (11Z,14Z)-11,14- Eicosadienoic acid

Mead acid (C20:3) (5Z,8Z,11Z)-5,8,11- Eicosatrienoic acid

Dihomo-γ- linolenic acid (C20:3) (8Z,11Z,14Z)-8,11,14- Eicosatrienoicacid

ω-3- Eicosatrienoic acid (C20:3) (11Z,14Z,17Z)- 11,14,17-Eicosatrienoicacid

Arachidonic acid (C20:4) (5Z,8Z,11Z,14Z)- 5,8,11,14-Eicosatetraenoicacid

Juniperonic acid (C20:4) (8Z,11Z,14Z,17Z)- 8,11,14,17- Eicosatetraenoicacid

Eicosapentaeonic acid (C20:5) (5Z,8Z,11Z,14Z,17Z)- 5,8,11,14,17-Eicosapentaenoic acid

Heneicosylic acid (C21:0) Heneicosanoic acid

Behenic acid (C22:0) Docosanoic acid

Brassidic acid (C22:1) (E)-13-Docosenoic acid

Erucic acid (C22:1) (Z)-13-Docosenoic acid

Docosadienoic acid (C22:2) (13Z,16Z)-13,16- Docosadienoic acid

Adrenic acid (C22:4) 7Z,10Z,13Z,16Z- Docosatetraenoic acid

Osbond acid (C22:5) (4Z,7Z,10Z,13Z,16Z)- 4,7,10,13,16- Docosapentaenoicacid

Clupanodonic acid (C22:5) (7Z,10Z,13Z,16Z,19Z)- 7,10,13,16,19-Docosapentaenoic acid

Docosahexaenoic acid (C22:6) (4Z,7Z,10Z,13Z,16Z,19Z)- 4,7,10,13,16,19-Docosahexaenoic acid

Herring acid (C24:6) (6Z,9Z,12Z,15Z,18Z, 21Z)-6,9,12,15,18,21-Tetracosahexaenoic acid

Heneicosylic acid (C23:0) Heneicosanoic acid

Lignoceric acid (C24:0) Tetracosanoic acid

Nervonic acid (C24:1) (Z)-15-Tetracosenoic acid

Tetracosanol- pentaenoic acid (C24:5) (9Z,12Z,15Z,18Z,21Z)-9,12,15,18,21- Tetracosapentaenoic acid

Cerotic acid (C26:0) Hexacosanoic acid

It will be understood that in the event of any inconsistency between thecommon name, the systematic name, and the chemical structure in Table 1,all such compounds will be considered as embraced by the invention. Forexample, in the event of a discrepancy in the systematic name and thestructure provided, both the compound corresponding to the systematicname number and the compound corresponding to the structure will beconsidered within the scope of the invention.

In some embodiments, the compounds of formula I(a) and/or II(a) willinclude a group R₁, R₂, and R₆ independently selected from the lipids ofTable 1.

Pharmaceutical compositions comprising lipid mixtures which may beextracted from hen egg yolk are described herein. In some embodiments,the pharmaceutical compositions are derived (e.g. extracted, chemicallyreacted, isolated, separated, and/or combinations thereof) from hen eggyolk. However, it should be understood that, unless otherwise stated,the invention is not limited to any method of preparation. Accordingly,in some embodiments, the lipid mixture may be obtained “synthetically,”for example, by preparing blends of the constituent lipids, derived fromany source or made synthetically. Without limitation, BAP(+) and BAP(−)preparations from which the lipid extracts of the invention may bederived are available from AREKO Ltd. (Prague, CZ). BAP(+) may besynthesized from BAP(−) by the methods disclosed in CZ Pat No 282,139 toKara et al., CZ Pat No 275,396 to Kara et al., CZ Pat No 276,477 to Karaet al., each hereby incorporated by reference in their entirety andspecifically in relation to synthetic preparation of biologically activephospholipids. In some embodiments, the method of preparation willdefine the components of the BAP mixture. In some embodiments, the lipidmixture may be obtained synthetically or derived from an animal, plant,or microbial (e.g., bacterial) source, be substantially identical to thecompositions prepared from hen egg yolk (e.g., the composition will haveabout 80% or about 90% or about 95% or about 99% or 100% of the samecomponents) and/or the abundance of the chief components (i.e., thoseconstituting >5% by weight of the extract) will be within about 50%, orabout 25%, or about 15%, or about 10% by weight of the abundance of thesame constituents in the hen egg yolk extracts described herein.

The BAP(−) extract may be isolated and purified as disclosed inGladkowski, W, et al., J. Am. Oil Chem, 89 179-182, (2012), herebyincorporated by reference in its entirety. In some embodiments, hen eggyolk extracts are derived from hen egg yolk (e.g., available from SigmaAldrich, Mo.) by organic solvent (e.g., methanol, ethanol, propanol,isopropanol, butanol, dichloromethane, acetone, hexane, or combinationsthereof) extraction. The solvent may be polar, polar-protic, and/ornonpolar. Preferably, the extract is obtained by extraction of hen eggyolk with ethanol and/or butanol. In some embodiments, hen egg yolkextracts are purified by de-oiling (precipitating) with acetone. In someembodiments, egg yolk extracts are precipitated with chilled (e.g., lessthan 10° C. or less than 5° C.) acetone. In various embodiments of theinvention, hen egg yolk extracts are reacted with an activatedcarboxylic acid species (e.g. acid anhydrides, acid chlorides, etc.).The activated acid species is an activated derivative (e.g., acylhydride, acyl halide, etc.) of palmitic acid. The carboxylic acid maybe, for example, a C₂-C₃₀ acid having an aliphatic chain, such as aC₁₋₂₉ saturated alkyl chain. In some embodiments, the extracts arereacted with 1,1′Carbonyldiimidazole (CDI) and an acid, such as a C₄-C₂₆fatty acid (e.g., palmitic acid, stearic acid, eicosanoic acid,tetradecanoic acid, hexanoic acid, etc.). In some embodiments, thereacted extracts may then be separated by chromatography to isolatespecific mixtures of phospholipids. Isolation of specific phospholipidsmay be performed using, for example, chromatographic separation (e.g.,column chromatography, flash chromatography, liquid crystalchromatography, thin layer chromatography, medium pressurechromatography, liquid column chromatography, semi-preparativechromatography, silica gel chromatography, reverse phase chromatography,etc.). In some embodiments, the isolation may involve the combination oftwo or more chromatographic separations to isolate specific fractions ofbiophospholipds. A schematic illustration of such a process is shown inFIG. 1. Also provided herein are methods for extraction of biologicallyactive phospholipids from a mixture of phospholipids (e.g. hen egg yolk,chemically treated egg yolk, etc.) comprising isolating variousfractions of said mixture by medium pressure silica gel chromatography.Typically, medium pressure gel chromatography utilizes tighter packedseparation columns, higher flow rates, and more precise gradient controlthan other chromatographic techniques. Medium pressure chromatographictechniques may allow for the phospholipid mixture to minimize contacttime with silica which, in turn, minimizes the amount of degradation ofthe phospholipid mixture (e.g., by converting PCs in to LPCs).

Typically, the flow rate in a chromatographic experiment may be set bydetermining the minimum variance per unit of column length (e.g., bydetermining the minimum variance per unit length of a separation columnto the linear mobile phase velocity by considering the physical kinetic,and thermodynamic properties of the separation). Typically thismaximizes the efficiency of the chromatographic experiment. However, ithas surprisingly been found that using a flow rate below the optimalvalue will result in improvements in the isolation of fractions withless degradation of biophospholipids. In some embodiments, thechromatography is performed with a silica column and a flow rate that issuboptimal for said column size. The flow rate for may be less thanabout 80% (e.g., less than about 50%) of the optimal flow rate. In someembodiments, the chromatography is performed with one or more eluentscomprising chloroform. In some embodiments the chromatography isperformed with at least two different eluents. One of these two eluentsmay be basic and the other of said at least two eluents may alcoholic(i.e. the eluent may comprise alcohol). The eluent may comprise water,ammonium hydroxide, triethylamine, methanol, ethanol, propanol,dichloromethane, isopropanol, chloroform, hexanes, butanol, orcombinations thereof. In some embodiments, the fractions of isolatedbiophospholipids may be further purified by removal of any degradationproducts produced during the isolation. For example, such purificationmay be achieved by extraction with a solvent comprising chloroform. Insome embodiments, the mixture of biologically active phospholipids maybe produced by the medium pressure chromatographic separation andpurification as described in Example 2.

In some embodiments, the extracts will comprise one or morephosphatidylcholines (PC). PCs may produce beneficial effects with otherPCs with different sets of acyl functional groups at positions R₁ andR₂. The acyl functional group may comprise a monovalent hydrocarbonradical that is saturated or unsaturated. By unsaturated, it is meantthat the hydrocarbon contains one or more double bonds (e.g. two, three,four, five, six, etc.). The hydrocarbon may be branched or unbranched.The hydrocarbon may be a C₄-C₃₅ (e.g C₁₀-C₃₀, C₁₂-C₂₈, C₁₄-C₂₄, etc.)alkyl, alkenyl, alkynyl, aryl, or aralkyl monovalent radical. Eachdouble bond in the hydrocarbon may independently be in the cis or transconfigurations. The hydrocarbon may have all double bonds in the cisconfiguration. Some embodiments of the invention involve pharmaceuticalcompositions comprising one or more phosphatidylcholines having thestructure of formula I(a):

where R₁ and R₂ are independently hydrogen or an acyl group comprising ahydrocarbon chain. In some embodiments, the hydrocarbon chain is thecorresponding acyl group of a naturally occurring fatty acid. In someembodiments, the phosphatidylcholine is a lysophosphatidylcholine (e.g.,R₂ is hydrogen). In some embodiments, R₁ is hydrogen. In someembodiments, at least one of R₁ or R₂ is hydrogen. In some embodiments,neither R₁ nor R₂ is hydrogen. The hydrocarbon chain may be saturated orunsaturated. In some embodiments, one of R₁ and R₂ may be saturated andthe other of R₁ and R₂ may be unsaturated. In other embodiments, both R₁and R₂ are both saturated. In other embodiments, R₁ and R₂ areunsaturated. In some embodiments, each of R₁ and R₂ is independentlychosen from a fatty acid (or acyl portion thereof) listed in Table 1.The acyl group may be derived from a fatty acid with any ω number (i.e.,ω-3, ω-4, ω-5, ω-6, ω-8, etc.).

Table 2 provides a non-limiting list of phosphatidylcholines that may beused beneficially, alone or in combination with one another, forproviding a patient benefit, for example, cancer treatment.

TABLE 2 Compound Name R₁ R₂ PC1 1-palmitoleoyl-2-linoleoyl-sn-glycero-3-(C16:1) (C18:2) phosphocholine PC21-palmitoyl-2-α-linolenoyl-sn-glycero-3- (C16:0) (C18:3) phosphocholinePC3 1-palmitoyl-2-arachidonoyl-sn-glycero-3- (C16:0) (C20:0)phosphocholine PC4 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (C18:2)(C18:2) PC5 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3- (C16:0) (C20:6)phosphocholine PC6 1-α-linolenoyl-2-docosahexaenoyl-sn-glycero-3-(C18:3) (C20:6) phosphocholine PC7 1-palmitoleoyl-2-oleoyl-sn-glycero-3-(C16:1) (C18:1) phosphocholine PC8 1-palmitoyl-2-linoleoyl-sn-glycero-3-(C16:0) (C18:2) phosphocholine PC91-palmitoleoyl-2-docosahexaenoyl-sn-glycero-3- (C16:1) (C20:6)phosphocholine PC10 1-palmitoyl-2-osbondoyl-sn-glycero-3- (C16:0)(C22:5) phosphocholine PC11 1-oleoyl-2-arachidonoyl-sn-glycero-3-(C18:1) (C20:0) phosphocholine PC12 1-oleoyl-2-linoleoyl-sn-glycero-3-(C18:1) (C18:2) phosphocholine PC131-stearoyl-2-arachidonoyl-sn-glycero-3- (C18:0) (C20:0) phosphocholinePC14 1-palmitoyl-2-oleoyl-sn-glycero-3- (C16:0) (C18:1) phosphocholinePC15 1-oleoyl-2-docosahexaenoyl-sn-glycero-3- (C18:1) (C20:6)phosphocholine PC16 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1)(C18:1) PC17 1-stearoyl-2-linoleoyl-sn-glycero-3- (C18:0) (C18:2)phosphocholine PC18 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine(C18:0) (C18:1) PC19 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine(C16:1) (C16:1) PC20 1-palmitoleoyl-2-palmitoyl-sn-glycero-3- (C16:1)(C16:0) phosphocholine PC21 1-palmitoleoyl-2-α-linolenoyl-sn-glycero-3-(C16:1) (C18:3) phosphocholine PC221-palmitoleoyl-2-stearoyl-sn-glycero-3- (C16:1) (C18:0) phosphocholinePC23 1-palmitoleoyl-2-arachidonoyl-sn-glycero-3- (C16:1) (C20:0)phosphocholine PC24 1-palmitoleoyl-2-osbondoyl-sn-glycero-3- (C16:1)(C22:5) phosphocholine PC25 1-palmitoyl-2-palmitoleoyl-sn-glycero-3-(C16:0) (C16:1) phosphocholine PC261,2-dipalmitoyl-sn-glycero-3-phosphocholine (C16:0) (C16:0) PC271-palmitoyl-2-stearoyl-sn-glycero-3- (C16:0) (C18:0) phosphocholine PC281-linoleoyl-2-palmitoleoyl-sn-glycero-3- (C18:2) (C16:1) phosphocholinePC29 1-linoleoyl-2-palmitoyl-sn-glycero-3- (C18:2) (C16:0)phosphocholine PC30 1-linoleoyl-2-α-linolenoyl-sn-glycero-3- (C18:2)(C18:3) phosphocholine PC31 1-linoleoyl-2-oleoyl-sn-glycero-3- (C18:2)(C18:1) phosphocholine PC32 1-linoleoyl-2-stearoyl-sn-glycero-3- (C18:2)(C18:0) phosphocholine PC33 1-linoleoyl-2-arachidonoyl-sn-glycero-3-(C18:2) (C20:0) phosphocholine PC341-linoleoyl-2-docosahexaenoyl-sn-glycero-3- (C18:2) (C20:6)phosphocholine PC35 1-linoleoyl-2-osbondoyl-sn-glycero-3- (C18:2)(C22:5) phosphocholine PC36 1-α-linolenoyl-2-palmitoleoyl-sn-glycero-3-(C18:3) (C16:1) phosphocholine PC371-α-linolenoyl-2-palmitoyl-sn-glycero-3- (C18:3) (C16:0) phosphocholinePC38 1-α-linolenoyl-2-linoleoyl-sn-glycero-3- (C18:3) (C18:2)phosphocholine PC39 1,2-di-α-linolenoyl-sn-glycero-3-phosphocholine(C18:3) (C18:3) PC40 1-α-linolenoyl-2-oleoyl-sn-glycero-3- (C18:3)(C18:1) phosphocholine PC41 1-α-linolenoyl-2-stearoyl-sn-glycero-3-(C18:3) (C18:0) phosphocholine PC421-α-linolenoyl-2-arachidonoyl-sn-glycero-3- (C18:3) (C20:0)phosphocholine PC43 1-α-linolenoyl-2-osbondoyl-sn-glycero-3- (C18:3)(C22:5) phosphocholine PC44 1-oleoyl-2-palmitoleoyl-sn-glycero-3-(C18:1) (C16:1) phosphocholine PC45 1-oleoyl-2-palmitoyl-sn-glycero-3-(C18:1) (C16:0) phosphocholine PC461-oleoyl-2-α-linolenoyl-sn-glycero-3- (C18:1) (C18:3) phosphocholinePC47 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (C18:1) (C18:0)PC48 1-oleoyl-2-osbondoyl-sn-glycero-3- (C18:1) (C22:5) phosphocholinePC49 1-stearoyl-2-palmitoleoyl-sn-glycero-3- (C18:0) (C16:1)phosphocholine PC50 1-stearoyl-2-palmitoyl-sn-glycero-3- (C18:0) (C16:0)phosphocholine PC51 1-stearoyl-2-α-linolenoyl-sn-glycero-3- (C18:0)(C18:3) phosphocholine PC52 1,2-distearoyl-sn-glycero-3-phosphocholine(C18:0) (C18:0) PC53 1-stearoyl-2-docosahexaenoyl-sn-glycero-3- (C18:0)(C20:6) phosphocholine PC54 1-stearoyl-2-osbondoyl-sn-glycero-3- (C18:0)(C22:5) phosphocholine PC55 1-arachidonoyl-2-palmitoleoyl-sn-glycero-3-(C20:0) (C16:1) phosphocholine PC561-arachidonoyl-2-palmitoyl-sn-glycero-3- (C20:0) (C16:0) phosphocholinePC57 1-arachidonoyl-2-linoleoyl-sn-glycero-3- (C20:0) (C18:2)phosphocholine PC58 1-arachidonoyl-2-α-linolenoyl-sn-glycero-3- (C20:0)(C18:3) phosphocholine PC59 1-arachidonoyl-2-oleoyl-sn-glycero-3-(C20:0) (C18:1) phosphocholine PC601-arachidonoyl-2-stearoyl-sn-glycero-3- (C20:0) (C18:0) phosphocholinePC61 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (C20:0) (C20:0) PC621-arachidonoyl-2-docosahexaenoyl-sn-glycero-3- (C20:0) (C20:6)phosphocholine PC63 1-arachidonoyl-2-osbondoyl-sn-glycero-3- (C20:0)(C22:5) phosphocholine PC641-docosahexaenoyl-2-palmitoleoyl-sn-glycero-3- (C20:6) (C16:1)phosphocholine PC65 1-docosahexaenoyl-2-palmitoyl-sn-glycero-3- (C20:6)(C16:0) phosphocholine PC66 1-docosahexaenoyl-2-linoleoyl-sn-glycero-3-(C20:6) (C18:2) phosphocholine PC671-docosahexaenoyl-2-α-linolenoyl-sn-glycero-3- (C20:6) (C18:3)phosphocholine PC68 1-docosahexaenoyl-2-oleoyl-sn-glycero-3- (C20:6)(C18:1) phosphocholine PC69 1-docosahexaenoyl-2-stearoyl-sn-glycero-3-(C20:6) (C18:0) phosphocholine PC701-docosahexaenoyl-2-arachidonoyl-sn-glycero-3- (C20:6) (C20:0)phosphocholine PC71 1,2-didocosahexaenoyl-sn-glycero-3- (C20:6) (C20:6)phosphocholine PC72 1-docosahexaenoyl-2-docosahexaenoyl-sn- (C20:6)(C20:6) glycero-3-phosphocholine PC731-docosahexaenoyl-2-osbondoyl-sn-glycero-3- (C20:6) (C22:5)phosphocholine PC74 1-osbondoyl-2-palmitoleoyl-sn-glycero-3- (C22:5)(C16:1) phosphocholine PC75 1-osbondoyl-2-palmitoyl-sn-glycero-3-(C22:5) (C16:0) phosphocholine PC761-osbondoyl-2-linoleoyl-sn-glycero-3- (C22:5) (C18:2) phosphocholinePC77 1-osbondoyl-2-α-linolenoyl-sn-glycero-3- (C22:5) (C18:3)phosphocholine PC78 1-osbondoyl-2-oleoyl-sn-glycero-3- (C22:5) (C18:1)phosphocholine PC79 1-osbondoyl-2-stearoyl-sn-glycero-3- (C22:5) (C18:0)phosphocholine PC80 1-osbondoyl-2-arachidonoyl-sn-glycero-3- (C22:5)(C20:0) phosphocholine PC81 1-osbondoyl-2-docosahexaenoyl-sn-glycero-3-(C22:5) (C20:6) phosphocholine PC821,2-diosbondoyl-sn-glycero-3-phosphocholine (C22:5) (C22:5) PC831-eicosapentaenoyl-2-palmitoleoyl-sn-glycero-3- (C20:5) (C16:1)phosphocholine PC84 1-eicosapentaenoyl-2-palmitoyl-sn-glycero-3- (C20:5)(C16:0) phosphocholine PC85 1-eicosapentaenoyl-2-linoleoyl-sn-glycero-3-(C20:5) (C18:2) phosphocholine PC861-eicosapentaenoyl-2-α-linolenoyl-sn-glycero-3- (C20:5) (C18:3)phosphocholine PC87 1-eicosapentaenoyl-2-oleoyl-sn-glycero-3- (C20:5)(C18:1) phosphocholine PC88 1-eicosapentaenoyl-2-stearoyl-sn-glycero-3-(C20:5) (C18:0) phosphocholine PC891-eicosapentaenoyl-2-arachidonoyl-sn-glycero-3- (C20:5) (C20:0)phosphocholine PC90 1-eicosapentaenoyl-2-docosahexaenoyl-sn- (C20:5)(C20:6) glycero-3-phosphocholine PC911,2-dieicosapentaenoyl-sn-glycero-3- (C20:5) (C20:5) phosphocholine PC921-eicosapentaenoyl-2-osbondoyl-sn-glycero-3- (C20:5) (C22:5)phosphocholine PC93 1-eicosapentaenoyl-2-clupanodonoyl-sn-glycero-(C20:5) (C22:5) 3-phosphocholine PC941-palmitoyl-2-eicosapentaenoyl-sn-glycero-3- (C16:0) (C20:5)phosphocholine PC95 1-palmitoyl-2-clupanodonoyl-sn-glycero-3- (C16:0)(C22:5) phosphocholine PC96 1-linoleoyl-2-eicosapentaenoyl-sn-glycero-3-(C18:2) (C20:5) phosphocholine PC971-linoleoyl-2-clupanodonoyl-sn-glycero-3- (C18:2) (C22:5) phosphocholinePC98 1-α-linolenoyl-2-eicosapentaenoyl-sn-glycero-3- (C18:3) (C20:5)phosphocholine PC99 1-α-linolenoyl-2-clupanodonoyl-sn-glycero-3- (C18:3)(C22:5) phosphocholine PC100 1-oleoyl-2-eicosapentaenoyl-sn-glycero-3-(C18:1) (C20:5) phosphocholine PC1011-oleoyl-2-clupanodonoyl-sn-glycero-3- (C18:1) (C22:5) phosphocholinePC102 1-stearoyl-2-eicosapentaenoyl-sn-glycero-3- (C18:0) (C20:5)phosphocholine PC103 1-stearoyl-2-clupanodonoyl-sn-glycero-3- (C18:0)(C22:5) phosphocholine PC1041-arachidonoyl-2-eicosapentaenoyl-sn-glycero-3- (C20:0) (C20:5)phosphocholine PC105 1-arachidonoyl-2-clupanodonoyl-sn-glycero-3-(C20:0) (C22:5) phosphocholine PC1061-docosahexaenoyl-2-eicosapentaenoyl-sn- (C20:6) (C20:5)glycero-3-phosphocholine PC107 1,2-diclupanodonoyl-sn-glycero-3- (C22:5)(C22:5) phosphocholine PC1081-palmitoleoyl-2-eicosapentaenoyl-sn-glycero-3- (C16:1) (C20:5)phosphocholine PC109 1-palmitoleoyl-2-clupanodonoyl-sn-glycero-3-(C16:1) (C22:5) phosphocholine PC1101-docosahexaenoyl-2-clupanodonoyl-sn-glycero- (C20:6) (C22:5)3-phosphocholine PC111 1-osbondoyl-2-eicosapentaenoyl-sn-glycero-3-(C22:5) (C20:5) phosphocholine PC1121-osbondoyl-2-clupanodonoyl-sn-glycero-3- (C22:5) (C22:5) phosphocholinePC113 1-clupanodonoyl-2-eicosapentaenoyl-sn-glycero- (C22:5) (C20:5)3-phosphocholine PC114 1-palmitoleoyl-2-linoleoyl-sn-glycero-3- (C16:1)(C18:2) phosphocholine PC115 1-palmitoyl-2-α-linolenoyl-sn-glycero-3-(C16:0) (C18:3) phosphocholine PC1161-palmitoyl-2-arachidonoyl-sn-glycero-3- (C16:0) (C20:0) phosphocholinePC117 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (C18:2) (C18:2) PC1181-palmitoyl-2-docosahexaenoyl-sn-glycero-3- (C16:0) (C20:6)phosphocholine PC119 1-α-linolenoyl-2-docosahexaenoyl-sn-glycero-3-(C18:3) (C20:6) phosphocholine PC1201-palmitoleoyl-2-oleoyl-sn-glycero-3- (C16:1) (C18:1) phosphocholinePC121 1-palmitoyl-2-linoleoyl-sn-glycero-3- (C16:0) (C18:2)phosphocholine PC122 1-palmitoleoyl-2-docosahexaenoyl-sn-glycero-3-(C16:1) (C20:6) phosphocholine PC1231-palmitoyl-2-osbondoyl-sn-glycero-3- (C16:0) (C22:5) phosphocholinePC124 1-oleoyl-2-arachidonoyl-sn-glycero-3- (C18:1) (C20:0)phosphocholine PC125 1-oleoyl-2-linoleoyl-sn-glycero-3- (C18:1) (C18:2)phosphocholine PC126 1-stearoyl-2-arachidonoyl-sn-glycero-3- (C18:0)(C20:0) phosphocholine PC127 1-palmitoyl-2-oleoyl-sn-glycero-3- (C16:0)(C18:1) phosphocholine PC128 1-oleoyl-2-docosahexaenoyl-sn-glycero-3-(C18:1) (C20:6) phosphocholine PC1291,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1) (C18:1) PC1301-stearoyl-2-linoleoyl-sn-glycero-3- (C18:0) (C18:2) phosphocholinePC131 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (C18:0) (C18:1)PC132 1-tetradecanoyl-2-palmitoleoyl-sn-glycero-3- (C14:0) (C16:1)phosphocholine PC133 1-tetradecanoyl-2-palmitoyl-sn-glycero-3- (C14:0)(C16:0) phosphocholine PC134 1-tetradecanoyl-2-linoleoyl-sn-glycero-3-(C14:0) (C18:2) phosphocholine PC1351-tetradecanoyl-2-α-linolenoyl-sn-glycero-3- (C14:0) (C18:3)phosphocholine PC136 1-tetradecanoyl-2-oleoyl-sn-glycero-3- (C14:0)(C18:1) phosphocholine PC137 1-tetradecanoyl-2-stearoyl-sn-glycero-3-(C14:0) (C18:0) phosphocholine PC1381-tetradecanoyl-2-arachidonoyl-sn-glycero-3- (C14:0) (C20:0)phosphocholine PC139 1-tetradecanoyl-2-docosahexaenoyl-sn-glycero-3-(C14:0) (C20:6) phosphocholine PC1401-tetradecanoyl-2-docosahexaenoyl-sn-glycero-3- (C14:0) (C20:6)phosphocholine PC141 1-tetradecanoyl-2-eicosapentaenoyl-sn-glycero-3-(C14:0) (C20:5) phosphocholine PC1421-tetradecanoyl-2-arachidonoyl-sn-glycero-3- (C14:0) (C20:0)phosphocholine PC143 1-tetradecanoyl-2-eicosapentaenoyl-sn-glycero-3-(C14:0) (C20:5) phosphocholine PC1441-tetradecanoyl-2-osbondoyl-sn-glycero-3- (C14:0) (C22:5) phosphocholinePC145 1-tetradecanoyl-2-clupanodonoyl-sn-glycero-3- (C14:0) (C22:5)phosphocholine PC146 1-tetradecanoyl-2-hydroxy-sn-glycero-3- (C14:0) Hphosphocholine PC147 1-palmitoleoyl-2-tetradecanoyl-sn-glycero-3-(C16:1) (C14:0) phosphocholine PC1481-palmitoyl-2-tetradecanoyl-sn-glycero-3- (C16:0) (C14:0) phosphocholinePC149 1-linoleoyl-2-tetradecanoyl-sn-glycero-3- (C18:2) (C14:0)phosphocholine PC150 1-α-linolenoyl-2-tetradecanoyl-sn-glycero-3-(C18:3) (C14:0) phosphocholine PC1511-oleoyl-2-tetradecanoyl-sn-glycero-3- (C18:1) (C14:0) phosphocholinePC152 1-stearoyl-2-tetradecanoyl-sn-glycero-3- (C18:0) (C14:0)phosphocholine PC153 1-arachidonoyl-2-tetradecanoyl-sn-glycero-3-(C20:0) (C14:0) phosphocholine PC1541-docosahexaenoyl-2-tetradecanoyl-sn-glycero-3- (C20:6) (C14:0)phosphocholine PC155 1-eicosapentaenoyl-2-tetradecanoyl-sn-glycero-3-(C20:5) (C14:0) phosphocholine PC1561-arachidonoyl-2-tetradecanoyl-sn-glycero-3- (C20:0) (C14:0)phosphocholine PC157 1-eicosapentaenoyl-2-tetradecanoyl-sn-glycero-3-(C20:5) (C14:0) phosphocholine PC1581-osbondoyl-2-tetradecanoyl-sn-glycero-3- (C22:5) (C14:0) phosphocholinePC159 1-clupanodonoyl-2-tetradecanoyl-sn-glycero-3- (C22:5) (C14:0)phosphocholine PC160 1-hydroxy-2-tetradecanoyl-sn-glycero-3- H (C14:0)phosphocholine PC161 1-hydroxy-2-palmitoleoyl-sn-glycero-3- H (C16:1)phosphocholine PC162 1-hydroxy-2-palmitoyl-sn-glycero-3- H (C16:0)phosphocholine PC163 1-hydroxy-2-linoleoyl-sn-glycero-3- H (C18:2)phosphocholine PC164 1-hydroxy-2-α-linolenoyl-sn-glycero-3- H (C18:3)phosphocholine PC165 1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine H(C18:1) PC166 1-hydroxy-2-stearoyl-sn-glycero-3- H (C18:0)phosphocholine PC167 1-hydroxy-2-arachidonoyl-sn-glycero-3- H (C20:0)phosphocholine PC168 1-hydroxy-2-docosahexaenoyl-sn-glycero-3- H (C20:6)phosphocholine PC169 1-hydroxy-2-docosahexaenoyl-sn-glycero-3- H (C20:6)phosphocholine PC170 1-hydroxy-2-eicosapentaenoyl-sn-glycero-3- H(C20:5) phosphocholine PC171 1-hydroxy-2-arachidonoyl-sn-glycero-3- H(C20:0) phosphocholine PC172 1-hydroxy-2-eicosapentaenoyl-sn-glycero-3-H (C20:5) phosphocholine PC173 1-hydroxy-2-osbondoyl-sn-glycero-3- H(C22:5) phosphocholine PC174 1-palmitoleoyl-2-hydroxy-sn-glycero-3-(C16:1) H phosphocholine PC175 1-palmitoyl-2-hydroxy-sn-glycero-3-(C16:0) H phosphocholine PC176 1-linoleoyl-2-hydroxy-sn-glycero-3-(C18:2) H phosphocholine PC177 1-α-linolenoyl-2-hydroxy-sn-glycero-3-(C18:3) H phosphocholine PC1781-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (C18:1) H PC1791-stearoyl-2-hydroxy-sn-glycero-3- (C18:0) H phosphocholine PC1801-arachidonoyl-2-hydroxy-sn-glycero-3- (C20:0) H phosphocholine PC1811-docosahexaenoyl-2-hydroxy-sn-glycero-3- (C20:6) H phosphocholine PC1821-docosahexaenoyl-2-hydroxy-sn-glycero-3- (C20:6) H phosphocholine PC1831-eicosapentaenoyl-2-hydroxy-sn-glycero-3- (C20:5) H phosphocholinePC184 1-arachidonoyl-2-hydroxy-sn-glycero-3- (C20:0) H phosphocholinePC185 1-eicosapentaenoyl-2-hydroxy-sn-glycero-3- (C20:5) Hphosphocholine PC186 1-osbondoyl-2-hydroxy-sn-glycero-3- (C22:5) Hphosphocholine

It will be understood that in the event of any inconsistency between thefatty acid and compound name both the compound and the sphingomyelincomprising the fatty acid are disclosed in Table 2.

In various embodiments, any of the phosphatidylcholines listed in Table2 may comprise individually from about 1-100% (w/w) (e.g., 1-10% (w/w)or 10-20% (w/w) or 20-30% or 30-40% (w/w) or 40-50% (w/w) or 50-60%(w/w) or 60-70% (w/w) or 70-80% (w/w) or 80-90% (w/w) or 90-100% (w/w))of the phospholipid component (or the phosphatidylcholine component) ofthe pharmaceutical compositions. In some embodiments,phosphotidylcholine component comprises between about 1-25% PC7 byweight of the phospholipid component and/or between about 1-25% PC8 byweight of the phospholipid component and/or between about 1-25% PC14 byweight of the phospholipid component and/or between about 1-25% PC16 byweight of the phospholipid component and/or between about 1-25%. In someembodiments, the phospholipid component (or the phosphotidylcholinecomponent) of the pharmaceutical compositions of the invention may befree of any of the phosphotidylcholine compounds (PC1-PC186) listed inTable 2, or may be substantially free of such compounds, by which ismeant that a given phosphatidylcholine is present in such small amountsas to not have a benefit in the treatment of cancer at the given leveland in any event will be less than 2.5% (w/w) or less than 1% (w/w) orless than 0.5% (w/w) or less than 0.1% (w/w) based on the total weightof the phospholipid component (or of the phosphatidylcholine component).

The mixtures of compounds suitable for present invention may comprisehighly unsaturated fatty acid components in the PCs. In someembodiments, PC molecules comprising at least five or at least sixunsaturated bonds will constitute from about 10-100% (w/w) of the lipidcomponents.

In some embodiments of the invention, the pharmaceutical composition maycomprise a hen egg yolk extract, comprising a phosphatidylcholinecomponent; wherein at least 50% (w/w) of said phosphatidylcholinecomponent is a compound having the structure of formula I(a):

wherein R₁ is selected from the group consisting of palmitoyl (C16:0),(C18:2) (e.g., linoleoyl), (C18:3) (e.g., α-linolenoyl), (C16:1) (e.g.,palmitoleoyl), (C18:1) (e.g., oleoyl), and stearoyl (C18:0) radicals;and R₂ is selected from an acyl group corresponding to a naturallyoccurring fatty acid. In other embodiments, R₁ is selected from an acylgroup corresponding to a naturally occurring fatty acid and R₂ isselected from (C18:2) (e.g., linoleoyl), (C18:3) (e.g., α-linolenoyl),(C20:4) (e.g., arachidonoyl), (C18:2) (e.g., linoleoyl), (C22:6) (e.g.,docosahexaenoyl), (C18:1) (e.g., oleoyl), (C22:5) (e.g.,docosapentaenoyl), (C22:5) (e.g. clupanodonoyl), or (C18:5) (e.g.,eicosapentaenoyl) radicals.

In other embodiments of the invention the pharmaceutical composition maycomprise a hen egg yolk extract comprising a phosphatidylcholinecomponent; wherein at least 50% of said phosphatidylcholine component isselected from the group consisting of:

-   1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-α-linolenoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-linoleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,-   1-palmitoleoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-palmitoleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-eicosatrienoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-clupanodonoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-osbondoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,    and pharmaceutically acceptable salts thereof. In some embodiments,    the pharmaceutical composition may comprise a hen egg yolk extract    comprising a phosphatidylcholine component; wherein at least 50% of    said phosphatidylcholine component is selected from the group    consisting of: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,    1-palmitoleoyl-2-oleoyl-sn-glycero-3-phosphocholine,    1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,    1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine,    1,2-dioleoyl-sn-glycero-phosphocholine,    1-hydroxy-2-oleoyl-sn-glycero-3-phosphocholine, and pharmaceutically    acceptable salts thereof.

In some embodiments of the invention, the mixture comprisessphingomyelin (SPH) compounds. Sphingomyelins of the invention may havethe structure of formula II(a)

where R₆ can be any monovalent radical, but is typically an acyl groupcomprising an aliphatic hydrocarbon chains, and more typically is theacyl portion groups of the corresponding naturally occurring fattyacids. The acyl groups generally have the form —(C═O)—R, where R is aC₂-C₃₀ aliphatic alkyl or alkenyl chain. These acyl groups correspond tothe acyl portion of naturally occurring fatty acids such as ω-3 and ω-6fatty acids and given in Table 1. In some embodiments of the invention,the mixture of bioactive lipids comprises one or more PC compoundsand/or one or more SPH compounds. In some embodiments, the hen egg yolkextract is enriched with one or more PC compounds and/or one or more SPHcompounds. In other embodiments, the hen egg yolk extract is enrichedwith one or more SPH compounds. In other embodiments, the hen egg yolkextract is enriched with one or more PC compounds and one or more SPHcompounds.

In various embodiments, the pharmaceutical composition may comprise anegg yolk extract, wherein the egg yolk extract is enriched in one ormore PCs selected from the group of compounds having the structure offormula I(a):

where R₁ is selected from the group consisting of hydrogen, palmitoyl(C16:0), linoleoyl (C18:2), α-linolenoyl (C18:3), palmitoleoyl (C16:1),oleoyl (C18:1), and stearoyl (C18:0) radicals; and R₂ is selected fromthe group consisting of hydrogen, linoleoyl (C18:2), α-linolenoyl(C18:3), arachidonoyl (C20:4), linoleoyl (C18:2), docosahexaenoyl(C22:6), oleoyl (C18:1), osbondoyl (C22:5), and eicosapentaenoyl (C18:5)radicals; and/or wherein the egg yolk extract may be enriched in one ormore sphingomyelins selected from the group of compounds having thestructure:

where R₆ is palmitoyl, stearoyl, or oleoyl, or pharmaceuticallyacceptable salts thereof. In some embodiments, the pharmaceuticalcomposition may comprise a hen egg yolk extract, wherein the hen eggyolk extract may comprise a phosphatidylcholine component and asphingomyelin component; wherein at least 50% of saidphosphatidylcholine component may be selected from the group consistingof:

-   1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-α-linolenoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-linoleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,-   1-palmitoleoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-osbondoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-palmitoleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-clupanodonoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-linolenoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-eicosatrienoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-linolenoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine,-   1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine,-   1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine,-   1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and    pharmaceutically acceptable salts thereof.

In some embodiments, the sphingomyelin component is between about 0.01and about 50% by weight of the phospholipid composition (e.g., about 0.1to about 30% or about 1% to about 20% or about 1% to about 10%, etc.).In some embodiments, at least 50% (w/w) of the sphingomyelin componentmay comprise one or more sphingomyelins selected from the group ofN-palmitoyl-D-erythro-sphingosylphosphorylcholine,N-stearoyl-D-erythro-sphingosylphosphorylcholine, andN-oleoyl-D-erythro-sphingosylphosphorylcholine. In some embodiments, thesphingomyelin at least 50% (w/w) of the sphingomyelein componentcomprises N-palmitoyl-D-erythro-sphingosylphosphorylcholine. In someembodiments, the sphingomyelin component consists or consistsessentially of N-palmitoyl-D-erythro-sphingosylphosphorylcholine.

Table 3 provides the compound names for representatives ofsphingomyelins (SPH) comprising a (C18:1) backbone and the fatty acidresidue of each sphingomyelin which may be present in the egg yolkextract. In some embodiments, the SPH has a different fatty acidbackbone (e.g., those fatty acids listed in Table 1). These SPHcompounds may be present individually or in combination with oneanother, or in combination with one or more PC compounds, e.g., asdescribed in Table 2.

TABLE 3 Compound Compound Name Fatty Acid SPH1N-palmitoyl-D-erythro-sphingosylphosphorylcholine (C16:0) SPH2N-stearoyl-D-erythro-sphingosylphosphorylcholine (C18:0) SPH3N-oleoyl-D-erythro-sphingosylphosphorylcholine (C18:1) SPH4N-palmitoleoyl-D-erythro-sphingosylphosphorylcholine (C16:1) SPH5N-linoleoyl-D-erythro-sphingosylphosphorylcholine (C18:2) SPH6N-α-linolenoyl-D-erythro-sphingosylphosphorylcholine (C18:3) SPH7N-arachidonoyl-D-erythro-sphingosylphosphorylcholine (C20:4) SPH8N-docosahexaenoyl-D-erythro-sphingosylphosphorylcholine (C22:6) SPH9N-eicosapentaenoyl-D-erythro-sphingosylphosphorylcholine (C20:5) SPH10N-osbondoyl-D-erythro-sphingosylphosphorylcholine (C22:5) SPH11N-clupanodonoyl-D-erythro-sphingosylphosphorylcholine (C22:5) SPH12N-tetradecanoyl-D-erythro-sphingosylphosphorylcholine (C14:0) SPH13N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (C17:0) SPH14N-nonadecanoyl-D-erythro-sphingosylphosphorylcholine (C19:0) SPH15N-eicosanoyl-D-erythro-sphingosylphosphorylcholine (C20:0) SPH16N-docosanoyl-D-erythro-sphingosylphosphorylcholine (C22:0) SPH17N-docosenoyl-D-erythro-sphingosylphosphorylcholine (C22:1) SPH18N-heneicosanoyl-D-erythro-sphingosylphosphorylcholine (C23:0) SPH19N-tetracosadienoic-D-erythro-sphingosylphosphorylcholine (C24:2) SPH20N-tetracosenoyl-D-erythro-sphingosylphosphorylcholine (C24:1) SPH21N-tetracosanoyl-D-erythro-sphingosylphosphorylcholine (C24:0)

It will be understood that in the event of any inconsistency between thefatty acid and compound name both the compound and the sphingomyelincomprising the fatty acid are disclosed in Table 3. In some embodiments,any combination of sphingomyelins selected from SPH1, SPH2, andSPH12-SPH21 may be present in the egg yolk extract as disclosed in Hsu,F. et al., J. Am. Soc. Mass. Spectrom. 11 2000 (437-449) hereinincorporated by reference in its entirety, particularly in reference todisclosure of egg yolk components.

In various embodiments, any of the sphingomyelins listed in Table 3 maycomprise individually from about 1-100% (w/w) (e.g., 1-10% (w/w) or10-20% (w/w) or 20-30% or 30-40% (w/w) or 40-50% (w/w) or 50-60% (w/w)or 60-70% (w/w) or 70-80% (w/w) or 80-90% (w/w) or 90-100% (w/w)) of thephospholipid component (or the sphingomyelin component) of thepharmaceutical compositions. In some embodiments, the sphingomyelincomponent comprises from about 0.01% to about 50% or about 0.1% to about10% by weight of the phospholipid component (e.g., 0.1 to about 30,about 1% to about 5% by weight of the phospholipid component). In someembodiments, the phospholipid component (or the sphingomyelin component)of the pharmaceutical compositions of the invention may be free of anyof sphingomyelin compounds (e.g. SPH1-SPH21 listed in Table 3), or maybe substantially free of such compounds by which is meant that a givensphingomyelin is present in such small amounts as to not have a benefitin the treatment of cancer at the given level and in any event will beless than 2.5% (w/w) or less than 1% (w/w) or less than 0.5% (w/w) orless than 0.1% (w/w) based on the total weight of the phospholipidcomponent (or of the sphingomyelin component).

In various embodiments, the pharmaceutical compositions may include oneor more (e.g., two or more, three or more, four or more, etc.)phosphatidylcholines selected from the group consisting of PC1-PC186(e.g., the group consisting of PC1-PC131) and/or one or moresphingomyelins (e.g., two or more, three or more, four or more, etc.)selected from the group consisting of SPH1-SPH11. In other embodiments,the pharmaceutical compositions may include one or morephosphatidylcholines selected from the group consisting of PC1-PC18and/or one or more sphingomyelins selected from the group consisting ofSPH1-SPH3. In other embodiments, the pharmaceutical compositions mayinclude two or more (e.g, three or more, four or more, five or more,etc.) phosphatidylcholines independently selected from the groupconsisting of PC1-PC18. In other embodiments, the pharmaceuticalcompositions may include one or more phosphatidylcholines (e.g., two ormore, three or more, four or more, etc.) and one or more sphingomyelins(e.g., two or more, three or more, four or more, etc.), where the one ormore phosphatidylcholines are selected from group consisting of PC1-PC18and the one or more sphingomyelins are selected from the groupconsisting of SPH1-SPH3. In some embodiments, the phosphatidylcholinecomponent comprises PC7 and/or PC8 and/or PC14 and/or PC16 and/or PC17.In some embodiments, the sphingomyelin component comprises SPH1. IN someembodiments, the sphingomyelin component consists or consistsessentially of SPH1.

In some embodiments, the pharmaceutical composition may include PC1 andone or more phosphatidylcholines selected from the group consisting ofPC2-PC18. In some embodiments, the pharmaceutical composition mayinclude PC2 and one or more phosphatidylcholines selected from the groupconsisting of PC1, and PC3-PC18. In some embodiments, the pharmaceuticalcomposition may include PC3 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC2, and PC4-PC18. In someembodiments, the pharmaceutical composition may include PC4 and one ormore phosphatidylcholines selected from the group consisting of PC1-PC3,and PC5-PC18. In some embodiments, the pharmaceutical composition mayinclude PC5 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC4, and PC6-PC18. In some embodiments, thepharmaceutical composition may include PC6 and one or morephosphatidylcholines selected from the group consisting of PC1-PC5, andPC7-PC18. In some embodiments, the pharmaceutical composition mayinclude PC7 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC6, and PC8-PC18. In some embodiments, thepharmaceutical composition may include PC8 and one or morephosphatidylcholines selected from the group consisting of PC1-PC7, andPC9-PC18. In some embodiments, the pharmaceutical composition mayinclude PC9 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC8, and PC10-PC18. In some embodiments, thepharmaceutical composition may include PC10 and one or morephosphatidylcholines selected from the group consisting of PC1-PC9, andPC11-PC18. In some embodiments, the pharmaceutical composition mayinclude PC11 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC10, and PC12-PC18. In some embodiments, thepharmaceutical composition may include PC12 and one or morephosphatidylcholines selected from the group consisting of PC1-PC11, andPC13-PC18. In some embodiments, the pharmaceutical composition mayinclude PC13 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC12, and PC14-PC18. In some embodiments, thepharmaceutical composition may include PC14 and one or morephosphatidylcholines selected from the group consisting of PC1-PC13, andPC15-PC18. In some embodiments, the pharmaceutical composition mayinclude PC15 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC14, and PC16-PC18. In some embodiments, thepharmaceutical composition may include PC16 and one or morephosphatidylcholines selected from the group consisting of PC1-PC15, andPC17-PC18. In some embodiments, the pharmaceutical composition mayinclude PC17 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC16, and PC18. In some embodiments, thepharmaceutical composition may include PC18 and one or morephosphatidylcholines selected from the group consisting of PC1-PC17.

In some embodiments, the pharmaceutical composition may include SPH1 andone or more phosphatidylcholines selected from the group consisting ofPC1-PC18. In some embodiments, the pharmaceutical composition mayinclude SPH2 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC18. In some embodiments, the pharmaceuticalcomposition may include SPH3 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC18.

In some implementations of the invention, the phospholipid mixtures areenriched in one or more phospholipids (e.g., phosphatidylcholines,sphingomyelins, lysophosphatidylcholines, etc.). By “enriched” is meantthat the relative abundance of a given phospholipid in relation to anyother phospholipids in the composition is increased compared to therelative abundance of the same phospholipid to the same otherphospholipids in hen egg yolk (or in BAP(−) or in BAP(+) or any fractionthereof).

In various embodiments, the hen egg yolk may be enriched in one or morephosphatidylcholines selected from the group consisting of PC1-PC186(e.g., the group consisting of PC1-PC131) and/or one or moresphingomyelins selected from the group consisting of SPH1-SPH11. Inother embodiments, the hen egg yolk is enriched in one or morephosphatidylcholines selected from the group consisting of PC1-PC18and/or one or more sphingomyelins selected from the group consisting ofSPH1-SPH3. In other embodiments, the hen egg yolk is enriched in two ormore phosphatidylcholines independently selected from the groupconsisting of PC1-PC18. In other embodiments, the hen egg yolk isenriched in one or more phosphatidylcholines and with one or moresphingomyelins, where the one or more phosphatidylcholines are selectedfrom group consisting of PC1-PC18 and the one or more sphingomyelins areselected from the group consisting of SPH1-SPH3.

In some embodiments, the hen egg yolk extract is enriched in PC1 and oneor more phosphatidylcholines selected from the group consisting ofPC2-PC18. In some embodiments, the hen egg yolk extract is enriched inPC2 and one or more phosphatidylcholines selected from the groupconsisting of PC1, and PC3-PC18. In some embodiments, the hen egg yolkextract is enriched in PC3 and one or more phosphatidylcholines selectedfrom the group consisting of PC1-PC2, and PC4-PC18. In some embodiments,the hen egg yolk extract is enriched in PC4 and one or morephosphatidylcholines selected from the group consisting of PC1-PC3, andPC5-PC18. In some embodiments, the hen egg yolk extract is enriched inPC5 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC4, and PC6-PC18. In some embodiments, the hen eggyolk extract is enriched in PC6 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC5, and PC7-PC18. In someembodiments, the hen egg yolk extract is enriched in PC7 and one or morephosphatidylcholines selected from the group consisting of PC1-PC6, andPC8-PC18. In some embodiments, the hen egg yolk extract is enriched inPC8 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC7, and PC9-PC18. In some embodiments, the hen eggyolk extract is enriched in PC9 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC8, and PC10-PC18. In someembodiments, the hen egg yolk extract is enriched in PC10 and one ormore phosphatidylcholines selected from the group consisting of PC1-PC9,and PC11-PC18. In some embodiments, the hen egg yolk extract is enrichedin PC11 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC10, and PC12-PC18. In some embodiments, the hen eggyolk extract is enriched in PC12 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC11, and PC13-PC18. In someembodiments, the hen egg yolk extract is enriched in PC13 and one ormore phosphatidylcholines selected from the group consisting ofPC1-PC12, and PC14-PC18. In some embodiments, the hen egg yolk extractis enriched in PC14 and one or more phosphatidylcholines selected fromthe group consisting of PC1-PC13, and PC15-PC18. In some embodiments,the hen egg yolk extract is enriched in PC15 and one or morephosphatidylcholines selected from the group consisting of PC1-PC14, andPC16-PC18. In some embodiments, the hen egg yolk extract is enriched inPC16 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC15, and PC17-PC18. In some embodiments, the hen eggyolk extract is enriched in PC17 and one or more phosphatidylcholinesselected from the group consisting of PC1-PC16, and PC18. In someembodiments, the hen egg yolk extract is enriched in PC18 and one ormore phosphatidylcholines selected from the group consisting ofPC1-PC17.

In some embodiments, the hen egg yolk extract is enriched in SPH1 andone or more phosphatidylcholines selected from the group consisting ofPC1-PC18. In some embodiments, the hen egg yolk extract is enriched inSPH2 and one or more phosphatidylcholines selected from the groupconsisting of PC1-PC18. In some embodiments, the hen egg yolk extract isenriched in SPH3 and one or more phosphatidylcholines selected from thegroup consisting of PC1-PC18.

Specific phospholipids according to the invention are illustrated below.

(R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC1”)

(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC2”)

(R)-2((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC3”)

(R)-2,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC4”)

(R)-2-(((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC5”)

(R)-2((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC6”)

(R)-2((7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC7”)

(R)-2((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC8”)

(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC9”)

(R)-2-((Z)-octadeca-9-enoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC10”)

(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC11”)

(R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC12”)

(R)-2-(((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC13”)

(R)-2-(oleoyloxy)-3-(palmitoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate (“MPC14”)

(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate (“MPC15”)

(R)-2,3-bis(oleoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate(“MPC16”)

(R)-2-(oleoyloxy)-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate (“MPC17”)

(2 S,3R,E)-2-heptadecanamido-3-hydroxyoctadec-4-en-1-yl(2-(trimethylammonio)ethyl) phosphate (“SPH1”)

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC1 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC1 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC2 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC2 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC3 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC3 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC4 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC4 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC5 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC5 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC6 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC6 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC7 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC7 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC8 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC8 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC9 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC9 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC10 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC10 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC11 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC11 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC12 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC12 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC13 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC13 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC14 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC14 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC15 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC15 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC16 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC16 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

In one embodiment, a pharmaceutical composition is provided comprising aphosphatidylcholine comprising, consisting essentially of, or consistingof an effective amount of MPC17 and a sphingomyelin comprising,consisting essentially of, or consisting of an effective amount of SPH1,together with a pharmaceutically acceptable carrier and/or excipient. Inone embodiment, the MPC17 and SPH1 exhibit enhanced activity incombinations composed to their individual activities. In one embodiment,the pharmaceutical composition is in the form of an oral dosage form(e.g., a capsule), as a fixed-dose combination.

Natural hen egg yolk extracts like BAP(−) may also containlysophosphatidylcholines (LPC) which are a class of phosphatidylcholineshaving the structure of formula III(a):

where R₁₃ can be any monovalent radical, but is more typically analiphatic hydrocarbon chain, and are even more typically the acylfunctional groups of naturally occurring fatty acids. Pharmaceuticalcompositions are also provided that are substantially free of thesecompounds. In some embodiments, the pharmaceutical composition may besubstantially free of some or each lysophosphatidylcholine (LPC) havingthe structure of formula III(a). However, in other embodiments, thepharmaceutical composition may comprise compounds having formula III(a).In some embodiments, the pharmaceutical composition comprises one ormore egg yolk extracts enriched with one or more LPCs (e.g., LPC'shaving the structure of formula III(a)). In some embodiments, R₁₃ ishydrogen or alkyl. In some embodiments, R₁₃ is an acyl group bonded to ahydrocarbon radical. In other embodiments, the pharmaceuticalcompositions may be substantially free of compounds and combinations ofcompounds having the structure of formula III where R₁₃ is hydrogen oran acyl group selected from palmitoyl, stearoyl, oleoyl, linoyl, orarachidonoyl. In other embodiments, the pharmaceutical compositionsfurther comprise compounds and combinations of compounds having thestructure of formula III(a) where R₁₃ is hydrogen or an acyl groupselected from palmitoyl, stearoyl, oleoyl, linoyl, or arachidonoyl. Inother embodiments, the composition is substantially free ofglycerophosphocholine.

Natural hen egg yolk extracts like BAP(−) may also containlysophosphatidylcholines (LPC) which are a class of phosphatidylcholineshaving the structure of formula III(b):

where R₁₃ can be any monovalent radical, but is more typically analiphatic hydrocarbon chain, and are even more typically the acylfunctional groups of naturally occurring fatty acids. Pharmaceuticalcompositions are also provided that are substantially free of thesecompounds. In some embodiments, the pharmaceutical composition may besubstantially free of some or each lysophosphatidylcholine (LPC) havingthe structure of formula III(a). However, in other embodiments, thepharmaceutical composition may comprise compounds having formula III(a).In some embodiments, the pharmaceutical composition comprises one ormore egg yolk extracts enriched with one or more LPCs (e.g., LPC'shaving the structure of formula III(a)). In some embodiments, R₁₃ ishydrogen or alkyl. In some embodiments, R₁₃ is an acyl group bonded to ahydrocarbon radical. In other embodiments, the pharmaceuticalcompositions may be substantially free of compounds and combinations ofcompounds having the structure of formula III where R₁₃ is hydrogen oran acyl group selected from palmitoyl, stearoyl, oleoyl, linoyl, orarachidonoyl. In other embodiments, the pharmaceutical compositionsfurther comprise compounds and combinations of compounds having thestructure of formula III(b) where R₁₃ is hydrogen or an acyl groupselected from palmitoyl, stearoyl, oleoyl, linoyl, or arachidonoyl. Inother embodiments, the composition is substantially free ofglycerophosphocholine.

Table 4 provides the compound names of certain lysophosphatidylcholines(LPC) which may be present in the hen egg yolk extract in someembodiments, and which may be excluded from the compositions in otherembodiments (or excluded in substantial amounts). These LPC compounds(e.g., those disclosed in Table 4) may be present individually or incombination with one another and/or in combination with one or more PCcompounds, e.g., as described in Table 2 and/or in combination with oneor move SPH compound, e.g., as described in Table 3. In someembodiments, the extract may be enriched by one or more LPC disclosed inTable 4 (e.g., LPC1, LPC2, LPC3, LPC4, LPCS, LPC6, LPC7, LPC8, LPC9, LPC10, LPC11, LPC12, or combinations thereof). It will be understood thatbecause LPCs are a class of PCs, an LPC may be referred to by either LPCnumber or the PC number.

TABLE 4 Compound Compound Name LPC1 glycerophosphocholine LPC22-palmitoyl-sn-glycero-3-phosphocholine LPC32-stearoyl-sn-glycero-3-phosphocholine LPC42-oleoyl-sn-glycero-3-phosphocholine LPC52-linoleoyl-sn-glycero-3-phosphocholine LPC62-arachidonoyl-sn-glycero-3-phosphocholine LPC72-α-linolenoyl-sn-glycero-3-phosphocholine LPC82-palmitoleoyl-sn-glycero-3-phosphocholine LPC92-docosahexaenoyl-sn-glycero-3-phosphocholine LPC102-eicosapentaenoyl-sn-glycero-3-phosphocholine LPC112-osbondoyl-sn-glycero-3-phosphocholine LPC122-clupanodoyl-sn-glycero-3-phosphocholine

In various embodiments, any of the LPC's listed in Table 4 may compriseindividually from about 1-100% (w/w) (e.g., 1-10% (w/w) or 10-20% (w/w)or 20-30% or 30-40% (w/w) or 40-50% (w/w) or 50-60% (w/w) or 60-70%(w/w) or 70-80% (w/w) or 80-90% (w/w) or 90-100% (w/w)) of thephospholipid component (or the lysophosphatidylcholine component) of thepharmaceutical compositions. In some embodiments, the phospholipidcomponent (or the lysophosphatidylcholine component) of thepharmaceutical compositions of the invention may be free of any of thelysophosphatidylcholine compounds (LPC1-LPC12) listed in Table 4, or maybe substantially free of such compounds by which is meant that a givenLPC is present in such small amounts as to not have a benefit in thetreatment of cancer at the given level and in any event will be lessthan 2.5% (w/w) or less than 1% (w/w) or less than 0.5% (w/w) or lessthan 0.1% (w/w) based on the total weight of the phospholipid component(or of the LPC component).

In one embodiment, the bioactive lipid composition comprises less than5% by weight and/or molar mass of LPC's. In other embodiments, thebioactive lipid compositions comprise less than 4%, less than 3%, lessthan 2%, less than 1%, less than 0.5%, less than 0.25%, or less than0.1% by weight and/or molar mass of LPC's.

BAP(+) may be produced through the acylation of the nitrogen of variousphosphatidylethanolamines (PE) found in the hen egg yolk extract. ThesePE compounds have the structure of formula I:

where R₁ and R₂ are defined as above, and R₃-R₅ may be hydrogen or anymonovalent hydrocarbon radical. In some embodiments, R₃ is selected fromhydrogen, methyl or a C₂-C₁₈ acyl group, and R₄ and R₅ are independentlyselected from the group consisting of hydrogen or alkyl (e.g., methyl,ethyl, propyl, etc.), and when R₃ is acyl R₄ or R₅ is absent and thenitrogen bonded to the R₃ group is uncharged. For example, R₃ may besaturated or R₃ may be the acyl group of a fatty acid. In someembodiments, the optionally N-acylated PC lipid may have an R₃ groupselected from myristoyl, palmitoyl, stearoyl, eicosenoyl, behenoyl, orlignoceroyl. In some embodiments, R₃ may be palmitoyl. In someembodiments, R₃, R₄ and R₅ are methyl.

In other embodiments, the pharmaceutical composition comprisesoptionally N-acylated lysophosphocholines. Optionally N-acylatedlysophosphoethanolamine are compounds having the structure of formulaIII:

where R₁₃ is defined as above, and R₁₀-R₁₂ may be hydrogen or anymonovalent hydrocarbon radical. In some embodiments, R₁₀ is selectedfrom hydrogen, methyl or a C₂-C₁₈ acyl group, and R₁₁ and R₁₂ areindependently selected from the group consisting of hydrogen or methyland in the case where R₁₀ is acyl, R₁₁ or R₁₂ is absent and the nitrogenbonded to R₁₀ is uncharged. For example, R₁₀ may be saturated or R₁₀ maybe the acyl group of a fatty acid. In some embodiments, R₁₀ ispalmitoyl. In some embodiments of the invention, a beneficial resultoccurs through enrichment of optionally N-acylated lysophosphocholine inegg yolk extract. In some embodiments, the pharmaceutical compositionmay further comprise an optionally N-acylated lysophosphocholine. Insome embodiments, the optionally N-acylated lysophosphocholine may havean R₁₀ group selected from myristoyl, palmitoyl, stearoyl, eicosenoyl,behenoyl, or lignoceroyl. In some embodiments, R₁₀ may be palmitoyl. Insome embodiments, R₁₀, R₁₁ and R₁₂ are methyl.

The pharmaceutical composition can be administered in any of a number ofways, including oral, parenteral, intravenous, systemic, local,intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation,or any such method of delivery. The pharmaceutical compositions may beadministered intravenously by injection. In one embodiment, a patient isgiven an intravenous infusion of a solution containing the PCs and/orSPHs through a running intravenous line over, for example, about 30minutes, about 60 minutes, about 90 minutes or longer. Suitableformulations carriers, diluents, and excipients can be found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., hereby incorporated by reference. Moreover, thepharmaceutical composition may be combined with another treatment forcancer in order to produce a beneficial effect between the two treatmentregimens.

The pharmaceutical composition may be formulated for oraladministration. In some embodiments, the pharmaceutical compositions maybe administered orally or parenterally in a suitable dosage form. Whenadministered orally, the pharmaceutical compositions described hereinmay be administered in the form of a tablet, gel capsule, liquidcapsule, emulsion or liquid. The formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques. Such techniques may include the step ofmixing the active ingredients and the pharmaceutical carrier(s),excipient(s), and/or diluent(s). In general, the formulations may beprepared by uniformly mixing the active ingredients with liquid carriersor finely divided solid carriers or both, and then, if necessary,shaping the product into, for example, a tablet form or capsule form. Inaddition, the pharmaceutical compositions may be incorporated intobiodegradable polymers allowing for sustained release of the compound,the polymers being implanted in the vicinity of where drug delivery isdesired, for example, at the site of a tumor or implanted so that thepharmaceutical compositions is released systematically.

In some embodiments, the compositions described herein may be formulatedas a liquid for oral administration. Liquid compositions includesolutions, suspensions and emulsions. Examples of liquid pharmaceuticalpreparations include propylene glycol solutions and solutions containingsweeteners for oral solutions, suspensions and emulsions.

In some embodiments, the unit dosage form is a capsule, such as a gelcapsule. In other embodiments, the compositions described herein can beformulated as a fill material for a capsule (e.g., a soft gelatincapsule). A capsule may be prepared, for example, by placing thecompositions described above inside a capsule shell. In some embodimentsof the invention, the compositions described herein may be filled intosoft capsules. A capsule shell may be made of methylcellulose,hydroxypropylmethylcellulose, polyvinyl alcohols, or denatured gelatinsor starch or other materials. In some embodiments, the compositions maybe filled in hard shell capsules. Hard shell capsules are typically madeof blends of relatively high gel strength bone and pork skin gelatins.Other suitable capsule shell materials include polyethylene,polypropylene, poly(methylmethacrylate), polyvinylchloride, polystyrene,polyurethanes, polytetrafluoroethylene, nylons, polyformaldehydes,polyesters, cellulose acetate, and nitrocellulose. The capsule shellitself may contain small amounts of dyes, opaquing agents, plasticizers,and preservatives.

The unit dosage form may also contain binders such as gum tragacanth,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, or alginicacid; and/or lubricants such as magnesium stearate. A sweetening agentsuch as sucrose, fructose, lactose or aspartame; or a flavoring agentsuch as peppermint, oil of wintergreen, or cherry flavoring, may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially nontoxic in the amounts employed. In addition, thephospholipids of the invention may be incorporated intosustained-release preparations and devices.

In various embodiments, the phospholipids of the invention can beadministered intravenously or intraperitoneally by infusion orinjection. Dispersions of the phospholipids can be prepared in water,optionally mixed with a nontoxic surfactant. Solutions can be preparedin glycerol, liquid polyethylene glycols, triacetin, or mixturesthereof, or in a pharmaceutically acceptable oil. Under ordinaryconditions of storage and use, preparations may contain one or moreantioxidants or preservatives, for example, to prevent the growth ofmicroorganisms.

In some embodiments of the invention, an effective (stabilizing) amountof one or more pharmaceutically acceptable anti-oxidants is added to theformulation. The term “anti-oxidant” is used herein to describe anycompound or combination of compounds that prevents or retards oxidation.Any of the known anti-oxidants may be used, including but not limited tobutylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate, lecithin, Vitamin E tocopherol, sesamin, sesamol, sesamolin,α-tocopherol, ascorbic acid, acorbyl palmitate, fumaric acid, malicacid, sodium ascorbate and sodium metabisulphite, as well as chelatingagents such as disodium EDTA, may also be used to stabilize theformulations of the present invention.

The amount of active compounds administered per dose is selected to beabove the minimal therapeutic dose but below a toxic dose. The choiceamount of dose will depend on a number of factors, such as the medicalhistory of the patient, the patient's age, the patient's weight, the useof other therapies, and the nature of the disease. The preferred doseand dosage regimen can be, for example, about 1 to about 20,000 mg orabout 10 to about 10,000 mg per kg body weight of the subject. Incertain embodiments, an initially low dose will be given, which can beincreased based on the response and/or tolerance of the patient to theinitial dose. Oral administration, for example, can occur at equalintervals, i.e., from about 1-10,000 mg/kg or from about 10 to about10,000 mg/kg every 24 hours (e.g., about 2.5 to about 250 mg/kg every 6hours) or every 12 hours or every 6 hours, etc. Dosing frequency may beonce daily, twice daily, or more frequent. Preferred unit dosageformulations are those containing a daily dose or unit, daily sub-dose,or an appropriate fraction thereof, of the administered ingredients. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the present invention may includeother agents conventional in the art having regard to the type offormulation in question.

In one embodiment, the pharmaceutical compositions of the invention areused to inhibit proliferative activity of neoplastic and in particularto treat neoplasms (e.g., cancers, pre-cancers, tumors, etc.). Thepharmaceutical compositions of the invention may be used to treatsarcoma and/or melanoma and/or carcinoma and/or lymphoma and/orleukemia.

The pharmaceutical compositions may be used for the treatment of cancer.In some embodiments, a method of treating bladder cancer in a mammal(e.g., a human) may comprise administration of a pharmaceuticalcomposition comprising an effective amount of a plurality of bioactivephospholipids according to the invention together with one or morepharmaceutically acceptable carriers and/or excipients. In someembodiments, a method of treating blood cancer may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In some embodiments, a method of treating brain(e.g., glioblastoma) cancer may comprise administration of apharmaceutical composition comprising an effective amount of a pluralityof bioactive phospholipids according to the invention together with oneor more pharmaceutically acceptable carriers and/or excipients. In someembodiments, a method of treating breast cancer may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In some embodiments, a method of treating cervicalcancer may comprise administration of a pharmaceutical compositioncomprising an effective amount of a plurality of bioactive phospholipidsaccording to the invention together with one or more pharmaceuticallyacceptable carriers and/or excipients. In some embodiments, a method oftreating colorectal cancer may comprise administration of apharmaceutical composition comprising an effective amount of a pluralityof bioactive phospholipids according to the invention together with oneor more pharmaceutically acceptable carriers and/or excipients. In someembodiments, a method of treating esophageal cancer may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In some embodiments, a method of treating kidneycancer may comprise administration of a pharmaceutical compositioncomprising an effective amount of a plurality of bioactive phospholipidsaccording to the invention together with one or more pharmaceuticallyacceptable carriers and/or excipients. In some embodiments, a method oftreating lung cancer may comprise administration of a pharmaceuticalcomposition comprising an effective amount of a plurality of bioactivephospholipids according to the invention together with one or morepharmaceutically acceptable carriers and/or excipients. In someembodiments, a method of treating ovarian cancer may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In some embodiments, a method of treating pancreaticcancer may comprise administration of a pharmaceutical compositioncomprising an effective amount of a plurality of bioactive phospholipidsaccording to the invention together with one or more pharmaceuticallyacceptable carriers and/or excipients. In some embodiments, a method oftreating skin cancer (e.g., melanoma or carcinoma) may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In some embodiments, a method of prostate bloodcancer may comprise administration of a pharmaceutical compositioncomprising an effective amount of a plurality of bioactive phospholipidsaccording to the invention together with one or more pharmaceuticallyacceptable carriers and/or excipients. In some embodiments, a method oftreating thyroid cancer may comprise administration of a pharmaceuticalcomposition comprising an effective amount of a plurality of bioactivephospholipids according to the invention together with one or morepharmaceutically acceptable carriers and/or excipients. In someembodiments, a method of treating uterine cancer may compriseadministration of a pharmaceutical composition comprising an effectiveamount of a plurality of bioactive phospholipids according to theinvention together with one or more pharmaceutically acceptable carriersand/or excipients. In each of the foregoing methods of treatment, theadministration may be oral, including by administration of a capsulecontaining said effective components of the bioactive phospholipidcompositions (e.g., MP1000) of the invention. In each of the foregoingmethods of treatment, the treatment is carried out for a time sufficientto shrink a tumor, slow progression of a cancer, affect remission of acancer, and/or inhibit proliferative activity of neoplasmatic cells.

EXAMPLES Example 1: Fractionation and Characterization of BAP(+) Mixture

A mixture of biologically active phospholipids (BAP(+)), extracted fromischemic chick embryonic tissue (hen egg yolk) with butanol, purified byacetone precipitation, and treated with palmitic acid/CDI was obtainedfrom AREKO Ltd., (Prague, Czech Republic). The BAP(+) mixture wasobtained as a 15% suspension in distilled water and maintained under N₂prior to use.

To begin separation of the complex BAP(+) mixture, the solvent wasremoved from BAP(+) by heating at 40° C. under reduced pressure.Following solvent removal, 3.0 g of the dried BAP(+) mixture wasseparated by flash chromatography using degassed solvents consisting ofdichloromethane, methanol, and water (8:2:0.1) eluent on a column packedwith, high-purity grade silica gel (60 Å pore size and 40-63 μm particlesize) from Sorbent Technologies (GA). The separation of BAP(+) throughthe column was monitored by thin layer chromatography (TLC) with aphosphomolybdic acid stain. FIG. 2A shows a representative separation ofeach of the fractions (F1-F5) which was obtained using achloroform:methanol:water (65:35:7 by volume) eluent and Meck KGA silicagel 60 F₂₅₄ plates as visualized by PMA stain. The first four fractions(F1-F4) eluted had retention factors (R_(f)) of 0.97, 0.90, 0.52, and0.28, respectively, as determined by TLC. Following complete elution offraction F4, the eluent was changed to a 6:4:0.6 dichloromethane,methanol and water mixture and the remainder of the BAP(+) material waseluted from column. This fraction is identified as fraction F5. Table 5illustrates the isolated amounts of each fraction and the approximate(%) abundance of each fraction in the BAP(+) mixture using the abovedescribed flash chromatographic fractionation technique. Spectroscopicanalysis of the fractions indicated that fraction F3 comprised NAEPEs.That PE's of BAP(−) are converted into NAEPE's can also be seen in FIG.2A. However, cytotoxicity studies of the fractions suggested thatfraction F5 provided the most cytotoxic activity to tumors (see, e.g.,Examples 6 and 7 below).

TABLE 5 % Abundance in Fraction Weight (mg) BAP(+) F1 402.9 13 F2 234.68 F3 505.0 17 F4 89.1 3 F5 1732.1 58

Example 2: Medium Pressure Chromatography of BAP Mixtures

Other fractionation techniques of BAP(+) were also employed in order toremove NAEPE's while minimizing the potential for decomposition of thePCs. It was determined that contact various BAP mixtures with silica mayresult in LPC formation and consequently an increase in LPC abundanceover time (e.g., presumably via degradation of PCs). The separation ofNAEPE containing fractions (e.g., fraction F3) from the otherbiologically active phospholipid fractions (e.g., F1, F2, F4 and F5)could be more effectively accomplished using medium pressure silica gelchromatography. Medium pressure silica gel chromatography was performedon a Combiflash® RF Lumen (available from Teledyne Isco, Nebr.). Theseparation was performed using a step gradient method of two eluentswhere the BAP(+) mixture is separated with a first eluent for a firstperiod of time followed immediately a second eluent for a second periodof time. For illustration purposes, separation of 10 g of BAP(+) mixturewas achieved using a 120 g Redisep Silica cartridge (cat #69-2203-320)with a flow of 43 ml/min. The mixture was first separated using a lineargradient that changed the ratio of one solvent (“Solvent A” or 0% B) toanother solvent (“Solvent B” or 100% B) over 25 minutes. After 25minutes, a 20-minute isocratic elution of 100% B was performed. SolventA was a chloroform:methanol:ammonium hydroxide mixture (8:2:0.05vol:vol:vol ratio) and solvent B was achloroform:methanol:water:ammonium hydroxide mixture (6:3.4:0.5:0.05vol:vol:vol ratio). Due to increased purity and better separation of thefractions produced using this chromatographic method, the isolatedamounts of each fraction was slightly different than for the flashchromatographic separation. Table 6 illustrates the isolated amounts ofeach fraction and the approximate (%) abundance of each fraction in theBAP(+) mixture using the medium pressure silica gel chromatographicfractionation technique.

TABLE 6 % Abundance in Fraction Weight (g) BAP(+) F1 + F2 1.65 18.4 F31.83 20.4 F4 0.385 5.43 F5 5.01 55.0 MP1000^(†) 4.85 54.0 ^(†)LPCsremoved from F5 with final chloroform extraction

Without wishing to be bound by theory, such a separation process allowsfor decreased elution time for the fraction thereby minimizing anydegradation products (e.g., LPCs) which may be produced while thefraction is in proximity with the column. For example, using mediumpressure chromatography, Fraction F5 may be isolated in about one hour,while flash chromatography conditions described in Example 1 may takeapproximately eight hours for separation. Successful isolation of thefractions was achieved by using a flow rate approximately 50% of theoptimum flow rate of the column used (e.g. the optimum flow rate atwhich there will be the minimum variance per unit of column length). Forexample, a 220 g cartridge has an optimal flow rate of about 150 mL/min.However, successful isolations occurred with flow rates of about 75mL/min. Such a trend was also found at 120 g and 80 g cartridges (whichhave an optimal flow rate of about 85 and 60 mL/min, respectively) but amore successful isolation of fraction F5 occurs (e.g., less degradation)with a flow rate on these columns of about 43 and 30 mL/minrespectively.

Further purification of Fraction F5 was achieved by extracting withchloroform:methanol:water (1:1:0.8 weight ratio) to remove residualglycerophosphocholine and other degradation products which may bepresent in the fraction. The resulting chloroform layer containing F5with LPC removed (“MP1000”) was collected and dried. Generally, betweenabout 12 and about 15 g of MP1000 may be separated from 30 g BAP(+)using this procedure. A chromatographic comparison of MP1000, BAP(+),SPH and LPC is illustrated in FIGS. 2B and 2C. The NAEPE componentseparated from BAP(+) is notably absent in fraction F5. Also, it can beseen that MP1000 is composed of fraction F5, but comprises asignificantly LPC component. Accordingly, MP1000 is a more than 90%purified fraction F5.

Example 3: Fractionation and Characterization of Fraction F5 and MP1000

Fraction F5 (and MP1000) were further fractionated into smaller groupsof lipids using preparative reverse phase chromatography on aThermoFisher UltiMate 3000 semipreparative HPLC system equipped with aflow splitter and a Corona Veo Charged Aerosol Detector (available fromDIONEX, CA) for sample detection. Preparative separation of lipidmixtures occurred on a HYPERSIL GOLD silica column (21×150 mm) with aflow rate of 5.0 mL/min with 30:70 eluent ratio (eluent A:eluent B byvolume) Eluent A was an acetonitrile:isopropanol solution (3:1 v/vratio) and eluent B was methanol:triethyl amine (0.9:0.1 v/v ratio).Fractions were collected, pooled and evaporated under reduced pressure.The resulting materials were dried under vacuum to constant weight foruse in spectroscopic, in vitro and/or in vivo experiments. Additionalsemi-preparative HPLC analysis was performed on Fraction F5 to in orderfurther isolate the fraction.

An HPLC chromatogram was taken of Fraction F5 (and MP1000) using a C18HYPERSIL GOLD silica column (250×10 mm, 4.5 mL/min) and isocraticconditions comprised of 30:70 A:B, where solvent A is acetonitrile+0.1%triethyl amine and solvent B is methanol+0.1% triethylamine. The HPLCchromatogram of MP1000 is shown in FIG. 3A. The fraction F5 producedaccording to Example 1 without application of additional pressure (i.e.,not using medium pressure chromatography) may also be separated intopeaks P1-P7. The HPLC chromatogram for fraction F5 isolated using theprotocol of Example 1 is illustrated in FIG. 3B. Table 7 gives therelative abundances of P1-P7 as measured in the chromatogram from FIG.3A.

TABLE 7 Sub- Approximate Fraction Relative Abundance P1 4.0% P2 3.2% P321.7% P4 3.5% P5 5.0% P6 49.3% P7 13.4%

Additional characterization of the constituents of sub-fractions P1 toP7 was performed using mass spectrometry and nuclear magnetic resonance(NMR) techniques. For mass spectrometry, samples of each fraction weredissolved in 5 mM ammonium acetate in methanol solution to aconcentration of 0.1 mg/mL. The components of each solution were ionizedusing an electrospray ionization (ESI) source and filtered by mass usinga quadrupole mass spectrometer (AB SCIEX 5000, available from AB SCIEX,MA) to detect the mass of each parent molecule in the fraction and therelative abundance of each parent molecule in the samples. The fattyacid residues were elucidated by neutral loss due to cationic ESI(ESI⁺). The parent molecule fragments to create the neutralcorresponding ketene of the fatty acid in ESI⁺. Conversely, anionic ESI(ESI⁻) results in anionic fatty acid loss from the corresponding core.Cross correlation of ESI⁺ and ESI⁻ mass spectroscopic results was usedto determine the position of the fatty acids in each core based on thefragmentation patterns of the parent molecules following ESI⁺ and ESI⁻as described in Hol{hacek over (c)}apeka, et.al., J. Chromatography A1218 (2011) 5146-5156, hereby incorporated by reference in its entirety.

Additionally, the sub-fractions were analyzed by the MS-MS techniques(University of Colorado Denver, Mass Spectrometry Lipidomics CoreFacility) disclosed in Jangle, R. D. et al. Ind. J. of Pharm. Sci. 75339-345 (2013), and Zacek, P. et al. J Lipid Res. 2016 December;57(12):2225-2234, each hereby incorporated by reference in theirentirety. Briefly, each sub-fraction was dissolved inmethanol:acetonitrile:water (60:20:20 vol:vol:vol ratio) with 1 mMammonium acetate and further mass spectroscopic analysis was appliedthereto. The molecular species were determined via infusion of eachsub-fraction solution into an AB Sciex triple quadrupole linear ion trapmass spectrometer at a flow rate of 10 μL/min. In positive ion mode, theorifice was set to +65V with a collision energy of 30V in order tomeasure a characteristic signal of phosphocholine phospholipids(phosphocholine ion with m/z=184). Collisional activation of eachmolecular species in negative ion mode allows for the determination offatty acids esterified to glycerol backbones. These collision inducedexperiments were performed with an electrospray voltage of −4800 V and ade-clustering potential of −120 V allowing for detection of the [M-15]⁻ions (parent loss of methyl anions). When compounds of the same mass arepresent in a mass spectrum, this protocol allows for the massspectroscopic detection of the predominant lipid contributing to m/zsignal at the same detected m/z peak.

Further characterization of the constituents within each fraction wasperformed using ¹H NMR spectroscopic, two dimensional correlationspectroscopic ([¹H-¹H]-2D-gCOSY) and heteronuclear single-quantumcorrelation spectroscopic ([¹H-¹³C]-gHSQC) techniques. Spectra wererecorded on a Varian Inova 500 instrument. The signal was analyzedrelative to the residual solvent peak for CDCl₃ (δ_(H)=7.26, andδ_(C)=77.4). The structures, as elucidated by mass spectrometry and NMRspectroscopy, of the molecular species of fractions P1 to P7 fractionare given in Table 8.

Fraction F5 comprises phosphatidylcholines (PC), sphingomyelin (SPH) andlow quantities of lysophosphatidylcholines (LPC). The LPC component ofF5 may comprise degradation products produced during the extraction andisolation of F5. A single fraction (P4) was identified to comprisesphingomyelins. Table 8 summarizes the major PC and LPC molecularspecies present in each other fraction of F5. Additionally, Table 8indicates the relative abundance of the molecular species (reported asmole percent) in each sub-fraction. Species comprising less than 5%(mole) are not shown. As shown in Table 8, the R₁, and R₂ functionalgroups of the PC species present in Fraction F5 correspond to acylgroups of the form —C(O)R where R is a saturated or unsaturatedhydrocarbon chain (e.g., palmitoyl is (C16:0)) or hydrogen. PC compoundshave the structure shown below in formula I(a):

TABLE 8 Lipid Mol. % in Lipid No. m/z R₁/R₂ (lipid number) fractionDesignation Fraction P1 1 806.6 (C16:0)/(C22:6) 61.6 MPC1  2* 756.6(C16:1)/(C18:2)- 15.1 MPC2 predominant and and MPC3 (C:16:0)/(C18:3)  3*782.7 (C18:2)/(C18:2)- 11.3 MPC4 predominant and and MPC5(C18:1)/(C18:3) Fraction P2 4 782.6 (C16:0)/(C20:4) 61.2 MPC6  5* 808.6(C16:0)/(C22:5) 18.2 MPC7 and and (C18:1)/(C20:4)- MPC8 predominantFraction P3  6* 758.7 (C16:0)/(C18:2)- 67.2 MPC9 predominant and andMPC10 (C16:1)/(C18:1) 7* 784.7 (C16:0)/(C20:3) 12.8 MPC11 and(C18:1)/(C18:2)- predominant 8 834.6 (C18:0)/(C22:6) 5.8 MPC12 FractionP4 (SPH component; SPH1) Fraction P5 9 810.6 (C18:0)/(C20:4) 67.3 MPC13Fraction P6 10  760.7 (C16:0)/C(18:1) 54.4 MPC14 11* 786.7(C18:0)/(C18:2)- 23.4 MPC15 predominant and and MPC16 (C18:1)/(C18:1)Fraction P7 12  886.6 (C18:0)/(C18:1) >90 MPC17 *The PC producing theindicated lipid number peaks may be either of the PCs indicated or acombination thereof. Generally, the lipid labeled “predominant” ispresent in mol percent of each indicated lipid number by more than 50%by mol.

Fraction P4 may not comprise PC compounds. However, Fraction P4comprises one or more SPH compounds having the structure of formulaII(a):

The sphingomyelin compound of Fraction P4 in Table 8 had a m/z of 703.8and was determined to have a palmitoyl acyl group (C16:0) at the R₆position (designated herein as “SPH1”).

Example 4: Separation of MP1000 into Lipid Classes Using Silica GelChromatography

MP1000 was separated into each lipid class (e.g., sphingomyelincomponent, phosphatidylcholine component, etc.) using normal phase HPLCusing a ThermoFisher Ultimate 3000 UPLC system equipped with a CoronaVeo RS Charged aerosol detector and a Hypersil GOLD Silica 150×4.6 mmcolumn, flow rate 1 ml/min, using isocratic conditions consisting of30:70 ratio of Eluent A=CHCl₃:MeOH:32% NH₃/H₂O solution (80:19.5:0.5 byvolume) and Eluent B=CHCl₃:MeOH:TEA:H₂O (69.53:25.58:0.49:4.40 byvolume). A representative chromatogram is shown in shown in FIG. 4. Ascan be seen, MP1000 predominantly comprises phosphatidylcholines andsphingomyelins (with a mol/mol ratio of 17.4:1).

Example 5: Fatty Acid Methyl Ester (FAME) Analysis of MP1000

The fatty acid groups of the phospholipids of MP1000 and a fraction from(BAP(−)) which was isolated using a method identical to that used forthe MP1000 isolation (“MP1000(−)”) were converted to methyl esters toidentify the fatty acid content in each fraction. Identification of thefatty acid content of the mixture of methyl esters was performed by gaschromatographic techniques to elucidate (FAME analysis by Matreya, LLCof State College, Pa.).

1 mL of 2% sulfuric acid in methanol (by volume) was added to 10 mg ofeach lipid mixture. The reaction mixtures were then heated for 30minutes at 80° C. and then cooled to room temperature. 0.5 mL of DIwater and 4 mL of hexane were then added. The reaction mixture wasshaken vigorously and left to stand until two phases became visible. Thehexane layer is collected. This hexane extraction is performed twoadditional times and pooled. A small amount of sodium sulfate:sodiumbicarbonate (4:1 ratio by weight) is added to the pooled hexane extract,shaken vigorously, and concentrated with a nitrogen stream to 0.5 mL toproduce the mixtures of methyl esters.

The mixtures of methyl esters were then analyzed using gaschromatography-flame ionization detection (GC-FID) which allows for theidentification of molar response factor of specific hydrocarbons. TheGC-FID device had an injection and detection temperatures of 250° C. TheGC-FID was run with a column comprising a non-bonded poly(80%biscyanopropyl/20% cyanopropylphenyl siloxane phase (SP-2330 Columnavailable from Supelco) dimensioned 30 m×0.25 mm×0.2 μm. The column wasinitially set to 170° C., the temperature was held for 17 minutes, thenchanged 10° C./min to 190° C. where the temperature was then held againfor 31 minutes. The GC-FID carrier gas had a linear velocity of 20cm/sec.

By comparing the retention times of authentic samples of fatty acidmethyl esters with the retention time of the FAME obtained from MP1000and MP1000(−) methyl ester mixtures allowed for identification of thefatty acid content in each fraction. FIG. 5 illustrates thechromatograms from the FAME analysis. The identified species areillustrated in Table 9. The hydrocarbon radical other than the methylgroup in each case is indicative of the fatty acid acyl fragment presentin the SPH's and PC's found in fraction F5. As can be seen, MP1000 andMP1000(−) appear to have similar fatty acid acyl content in thebiophospholipid mixture.

TABLE 9 % % Abundance Abundance in in Fatty acid species MP1000MP1000(−) Methyl hexadecanoate, (C16:0) 33 33 Methyl hexadecenoate,(C16:1, cis-9) 1 1 Methyl octadecanoate, (C18:0) 13 13 Methyloctadecenoate, (C18:1, cis-9) 30 31 Methyl octadecadienoate, (C18:2, allcis-9, 15 14 12) Methyl eicosatetraenoate, (C20:4, all cis-5, 4 3 8,11,14) Methyl docosahexaenoate, (C22:6, all cis-4, 2 1 7, 10,13,16,19)Methyl octadecatrienoate, (C18:3, all cis-9, >1 >1 12, 15) Methyldocosapentaenoate, (C22:5, all cis-7, >1 >1 10,13, 16,19)

Example 6: In Vitro Cytotoxicity Analysis of BAP(+) and Fractions F1-F5

The cytotoxic potential of various BAP mixtures was measured using anXTT cell viability assay as follows. Stock solutions of samples BAP+,Fraction F5, sub-fractions of Fraction F5 (e.g., sub-fraction P1, etc.),and a fraction from untreated egg yolk (BAP(−)) which was isolated usinga method identical to that used for the Fraction F5 isolation (“F5(−)”)were prepared by dissolving or suspending the same amount of eachmaterial in phosphate buffer saline (PBS) of physiological concentrationand pH of 7.2. Egg phospholipid (BAP(−)) (previously shown not topossess significant cytotoxic activity) was added as an emulsifier in anamount of 0.5% (w/w) for samples that did not form stable suspensions.Solutions/suspensions were prepared by vigorously shaking the componentsin vials under nitrogen atmosphere for 1-2 hours at ambient temperature.Immediately before testing, the solutions were again homogenized byshaking and then diluted to the appropriate concentration in the cellgrowth medium to form the test solutions.

A standard XTT assay was used to measure the half minimal inhibitoryconcentrations (IC₅₀) of each of the test materials on the humanpancreatic ductal adenocarcinoma cell line (Capan-2).

Flat-bottomed microplates with 96 wells each (available from NUNC,Denmark) were seeded with Capan-2 cells (6×10³ cells/well) in 200 μL ofcell growth media. Cells were incubated for 24 hours at 37° C. in ahumidified 5% CO₂/95% air atmosphere. After 24 hours, 50 μL of the testsolutions were added to the wells (in triplicate) to achieveconcentrations ranging from 0.0125-0.4% (w/w). Plates were thenincubated for an additional 72 hours. Following incubation, 150 μL ofsupernatant was discarded and a mixture of 25 μL of a 1 mg/mL dyesolution of2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide(XTT) and 7.5 μg/mL N-methyl dibenzopyrazine methyl sulfate (PMS) wasadded to each well. Microplates were incubated for another 4 hours inthe absence of light.

The number of adenocarcinoma cells in each well was determined bymeasuring absorbance at 450 nm with an INFINITE F50 absorbance readerequipped with Magellen for INFINITE F50 software (Tecan Austria GmbH,Austria). Cells cultivated in fresh medium were used as controls. Allreported IC₅₀ values are the mean of four independent experiments. Table10 gives the inhibitory activity of various BAP mixtures tested.

TABLE 10 BAP IC₅₀ Mixture (mg/mL) BAP(+) 0.74 F5(−) no activity F5 1.17P1 0.20 P2 0.23 P3 0.55 P4 0.29 P5 0.30 P6 no activity P7 no activity

As can be seen in Table 10, BAP(+) and fraction F5 are active againstcancer cells, however, F5(−) and some sub-fractions of F5 are not. Themost active fractions of F5 (in decreasing order) are P1, P2, and P4.Similar results may also be expected on H358 (lung), M14 (melanoma), 231(breast), Pancl (pancreas), HeLa (cervical), SK-OV-3 (ovarian), HepG2(liver), HCT116 (colon), T98G (glioblastoma multiforme), Jurkat (T celllymphoma), DU-145 (prostate), or A549 (lung) cell cultures.

Example 7: In Vivo Measurement of Cytotoxicity of BAP Mixtures

In vivo measurements of tumor growth inhibition in mice were conductedto determine efficacy of BAP(+), BAP(−), and fractions thereof. A humanpancreatic carcinoma cell line (Capan-2 available from EuropeanCollection of Cell Cultures, Salisbury UK) was cultivated at 37° C. in ahumidified 5% CO₂/95% air atmosphere using high glucose D-MEM mediumsupplemented with 20% fetal bovine serum, 2% penicillin/streptomycin and1.25% L-glutamine. Before application, cells were harvested bytrypsinisation for 30 minutes. Harvested Capan-2 cells were administeredsubcutaneously to each mouse as 1×10⁷ cells in a mixture with a basementmembrane preparation (BD MATRIGEL, available from I.T.A.-Interact,Prague CZ). Administration was in the rear right flank of Hsd:athymicnude-Fox nlnu mice (available from Anlab, Prague CZ). When the tumor ineach mouse reached a volume of about 0.103-0.122 cm³, the testing of thetumor growth inhibition on each mouse began by oral administration of0.1 mL of a 30% (w/w) solution of each listed component in a sunfloweroil carrier once a day for 42 days.

Table 12 gives the percentage of tumor weight growth inhibition (% WGI)as compared to the control for various BAP mixtures including BAP(−),BAP(+), pooled fractions F1-F4, F5, a fraction comprising a pooledcollection of the F5 sub-fractions enriched in more PCs comprisingsaturated fatty acids acyl residues (“F5A”), a fraction which comprisinga pooled collection of F5 sub-fractions enriched in more PCs comprisingunsaturated fatty acid acyl residues (“F5B”), and a fraction of BAP(+)possibly enriched in LPC's (“F5C”). Glycerophosphocholine (GPC) was alsoevaluated. The only BAP(+) fractions which comprised a sphingomyelincomponent were fractions F5 and F5B.

TABLE 11 Sample % WGI F5 85.7% BAP(+) 80.7% F1 + F2 + F3 + F4 78.3% F5A27.8% F5B 24.9% BAP(−) −8.8% GPC −41.9% F5C −109.2%

As can be seen in Table 11, the in vivo experiments demonstrate theinhibitory effect specific mixtures of BAP(+) have on tumor growth.Fraction F5 and BAP(+) are each highly active at inhibiting tumorgrowth. Moreover, fractions F5A and F5B alone have significantly lessinhibitory behavior on tumor growth than fraction F5 (which containsboth F5A and F5B). It is also observed that fraction F5A, a fractionenriched in unsaturated PCs, has comparable activity to fraction F5B, afraction enriched in saturated PCs and SPHs, but the combination of F5Aand F5B is evidently required for maximum efficacy. Fraction F5C (whichis believed to contain LPC's) more than doubled tumor size. It is alsonotable that BAP(−) did not inhibit tumor growth.

Additionally, in vivo experiments were performed on F5 fractionsisolated from the semi-preparative medium pressure chromatographymethodology of Example 2 (“MP1000”). Dosing solutions comprising 5% and30% by weight concentration were prepared in sunflower oil. Mice withtumors were orally administered 0.1 mL of the MP1000 solutions once perday for 42 days. FIG. 6 illustrates the results of these experiments. Ascan be seen, the MP1000 fraction reduced the tumor weight significantlymore than BAP(−) and control at each time point. A t-test was performedon data at each time point, analyzing statistical significance betweenthe treatments from the sunflower oil control and 5% by weight MP1000(“*” indicates p<0.05, “**” indicates p<0.01, “***” indicates p<0.001,and “****” indicates p<0.0001). It is notable, even though MP1000 andMP1000(−) have similar fatty acid content as demonstrated by FAMEanalysis, BAP(−), which comprises MP1000(−), does not reduce the tumorsize relative to control.

The inhibitory activity of MP1000 was investigated at 1% by weightconcentration and 5% by weight concentration in mouse xenografts ofcancer cell lines MiaPaca (pancreatic) and Panc-1 (pancreatic). As shownin FIGS. 7 and 8, both doses were significantly superior in retardingtumor growth as compared to control (sunflower oil). The dose responsefor the amount of MP1000 showed a trend towards significance after 20days in the case of MiaPaca tumors. Each of the MP1000 samples showedstatistical significance over the sunflower oil control as indicated(“*” indicates p<0.05, “**” indicates p<0.01, “***” indicates p<0.001,and “****” indicates p<0.0001).

Example 8: Kinase Inhibition

Inhibition of various kinases was measured using fluorescence resonanceenergy transfer (FRET) analysis. Briefly, a kinase is used to transferthe γ-phosphate of ATP to a single tyrosine, serine, or threonineresidue in a synthetic FRET-peptide in the presence of each testedfraction. A kinase inhibitor will prevent phosphorylation. FRET-peptidescomprise a donor fluorophore and an acceptor fluorophore, where theacceptor fluorophore will only undergo fluorescence from the excitationwavelength of the donor fluorophore due to resonant energy transferthrough the peptide. A development reagent is used to cleave theun-phosphorylated FRET-peptides. An uncleaved FRET-peptide (i.e.,phosphorylated peptide) will therefore undergo measurable FRET of bothacceptor and donor and be indicative of kinase inhibition. Cleavage ofthe FRET-peptide (i.e., un-phosphorylated peptides), however, disruptsFRET between donor and acceptor fluorophores on the FRET-peptide thusresulting in significantly less acceptor fluoroesence and is indicativeof little kinase inhibition. Because phosphorylation of FRET-peptidessuppresses cleavage by the development reagent, calculation of the ratioof donor emission to acceptor emission after excitation of the donorfluorophore at approximately 400 nm is used to quantitate reactionprogress. Low ratios indicate the FRET-peptide is phosphorylated (i.e.,little kinase inhibition) and high ratios indicate the FRET-peptide isphosphorylated (i.e., kinase inhibition). The percent phosphorylationcan therefore be calculated from the emission ratio based on controlsfor the maximum and minimum phosphorylation. The percent inhibition canbe calculated based on the percent phosphorylation of a sample with eachfraction as compared to the percent phosphorylation without eachfraction.

The kinase inhibitory activity of various 10 μM solutions of variousfractions was measured using the SELECTSCREEN Kinase Profiling Serviceavailable from Life Technologies. Unless otherwise indicated, theconcentration of ATP in each well is the Michaelis-Menten constant(K_(m) value-the concentration of ATP when the phosphorylation reactionvelocity is equal half of the maximal velocity for the reaction). Table13 shows the kinase inhibitory activity of the various fractions and thecorresponding Z′ values. Z′ is a measure of statistical effect size toassess the quality of the assay. Z′>0.5 is indicative measurementseparated from the background and a reliable assay result. The average %inhibition is representative of two independent measurements.

TABLE 12 Average % Fraction Name Kinase Tested Inhibition Z′ BAP(−) ABL1−2 0.84 BAP(−) ABL2 (Arg) 2 0.80 BAP(−) AKT1 (PKB alpha) 6 0.90 BAP(−)ALK 3 0.83 BAP(−) AURKB (Aurora B) 6 0.71 BAP(−) AURKC (Aurora C) 5 0.75BAP(−) AXL 4 0.81 BAP(−)* BRAF V599E −1 0.64 BAP(−)* BRAF −4 0.72 BAP(−)BTK 9 0.83 BAP(−) CDC42 BPA 1 0.70 (MRCKA) BAP(−) CDC42 BPB 8 0.57(MRCKB) BAP(−) CDK1/cyclin B 3 0.92 BAP(−) CDK2/cyclin A −1 0.84 BAP(−)CDK5/p25 1 0.66 BAP(−) CHEK1 (CHK1) 4 0.80 BAP(−) CHEK2 (CHK2) 10 0.84BAP(−) CSF1R (FMS) 3 0.79 BAP(−) CSNK1E (CK1 4 0.81 epsilon) BAP(−)CSNK2A1 (CK2 0 0.91 alpha 1) BAP(−) EGFR (ErbB1) 3 0.89 BAP(−) EPHA1 50.83 BAP(−) EPHA2 −3 0.82 BAP(−) EPHB2 4 0.91 BAP(−) EPHB4 −1 0.73BAP(−) ERBB2 (HER2) −1 0.83 BAP(−) ERBB4 (HER4) 0 0.79 BAP(−) FER 6 0.88BAP(−) FES (FPS) 1 0.77 BAP(−) FGFR1 −5 0.80 BAP(−) FGFR4 −1 0.90 BAP(−)FGR 12 0.80 BAP(−) FLT1 (VEGFR1) 2 0.90 BAP(−) FLT3 3 0.78 BAP(−) FLT4(VEGFR3) −3 0.85 BAP(−) FYN 16 0.81 BAP(−) HCK 6 0.91 BAP(−) IGF1R −90.91 BAP(−) IKBKB (IKK beta) 3 0.82 BAP(−) JAK1 −1 0.75 BAP(−) JAK2 JH1JH2 13 0.62 V617F BAP(−) JAK2 JH1 JH2 5 0.85 BAP(−) JAK3 8 0.81 BAP(−)KDR (VEGFR2) 5 0.77 BAP(−) KIT 6 0.72 BAP(−) LCK 7 0.76 BAP(−) LYN A 20.87 BAP(−)* MAP2K1 (MEK1) −6 0.79 BAP(−)* MAP3K8 (COT) 14 0.87 BAP(−)MAP4K4 (HGK) 9 0.72 BAP(−) MAPK14 (p38 −8 0.84 alpha) Direct BAP(−) MATK(HYL) −6 0.83 BAP(−) MST1R (RON) 3 0.78 BAP(−) MST4 2 0.73 BAP(−) NEK2 30.73 BAP(−) NTRK1 (TRKA) 4 0.76 BAP(−) PAK4 4 0.80 BAP(−) PDGFRA (PDGFR10 0.81 alpha) BAP(−) PDGFRB (PDGFR 5 0.88 beta) BAP(−) PDK1 Direct 90.75 BAP(−) PIM1 3 0.79 BAP(−) PLK1 4 0.85 BAP(−) PLK3 −14 0.84 BAP(−)PTK2 (FAK) 3 0.90 BAP(−) PTK2B (FAK2) 0 0.84 BAP(−) PTK6 (Brk) −3 0.82BAP(−)* RAF1 (cRAF) 15 0.81 Y340D Y341D BAP(−) RET 6 0.77 BAP(−) ROCK111 0.86 BAP(−) ROS1 2 0.83 BAP(−) RPS6KB1 6 0.78 (p70S6K) BAP(−) SGK(SGK1) 12 0.90 BAP(−) SYK −5 0.84 BAP(−) TBK1 3 0.78 BAP(−) TEK (Tie2) 30.80 BAP(−) TYRO3 (RSE) 2 0.92 BAP(−) YES1 −1 0.78 BAP(−) CDK7/cyclin −40.82 H/MNAT1 BAP(−) CDK9/cyclin T1 −3 0.92 BAP(−) GSG2 (Haspin) 2 0.83BAP(−) PIK3CG (p110 12 0.87 gamma) BAP(−) SPHK1 6 0.62 BAP(+) ABL1 150.84 BAP(+) ABL2 (Arg) 26 0.80 BAP(+) AKT1 (PKB alpha) 9 0.90 BAP(+) ALK−1 0.83 BAP(+) AURKB (Aurora B) 37 0.71 BAP(+) AURKC (Aurora C) 16 0.75BAP(+) AXL 22 0.81 BAP(+)* BRAF V599E 4 0.64 BAP(+)* BRAF −6 0.72 BAP(+)BTK 13 0.83 BAP(+) CDC42 BPA −4 0.70 (MRCKA) BAP(+) CDC42 BPB 2 0.57(MRCKB) BAP(+) CDK1/cyclin B 1 0.92 BAP(+) CDK2/cyclin A 3 0.84 BAP(+)CDK5/p25 18 0.66 BAP(+) CHEK1 (CHK1) −23 0.81 BAP(+) CHEK2 (CHK2) 5 0.84BAP(+) CSF1R (FMS) 0 0.79 BAP(+) CSNK1E (CK1 10 0.81 epsilon) BAP(+)CSNK2A1 (CK2 3 0.91 alpha 1) BAP(+) EGFR (ErbB1) 1 0.89 BAP(+) EPHA1 80.83 BAP(+) EPHA2 −1 0.82 BAP(+) EPHB2 5 0.91 BAP(+) EPHB4 2 0.73 BAP(+)ERBB2 (HER2) −12 0.86 BAP(+) ERBB4 (HER4) 5 0.79 BAP(+) FER 21 0.88BAP(+) FES (FPS) 3 0.77 BAP(+) FGFR1 2 0.80 BAP(+) FGFR4 15 0.90 BAP(+)FGR 29 0.80 BAP(+) FLT1 (VEGFR1) 7 0.90 BAP(+) FLT3 6 0.78 BAP(+) FLT4(VEGFR3) 5 0.85 BAP(+) FYN 30 0.81 BAP(+) HCK 7 0.91 BAP(+) IGF1R −40.91 BAP(+) IKBKB (IKK beta) 12 0.82 BAP(+) JAK1 −6 0.75 BAP(+) JAK2 JH1JH2 −5 0.62 V617F BAP(+) JAK2 JH1 JH2 −6 0.85 BAP(+) JAK3 13 0.81 BAP(+)KDR (VEGFR2) 5 0.77 BAP(+) KIT −1 0.72 BAP(+) LCK 20 0.76 BAP(+) LYN A 70.87 BAP(+)* MAP2K1 (MEK1) −4 0.79 BAP(+)* MAP3K8 (COT) 43 0.87 BAP(+)MAP4K4 (HGK) 11 0.72 BAP(+) MAPK14 (p38 −4 0.84 alpha) Direct BAP(+)MATK (HYL) −6 0.83 BAP(+) MST1R (RON) 2 0.78 BAP(+) MST4 3 0.73 BAP(+)NEK2 −10 0.71 BAP(+) NTRK1 (TRKA) −3 0.76 BAP(+) PAK4 7 0.80 BAP(+)PDGFRA (PDGFR −4 0.81 alpha) BAP(+) PDGFRB (PDGFR 3 0.88 beta) BAP(+)PDK1 Direct 2 0.75 BAP(+) PIM1 −4 0.79 BAP(+) PLK1 4 0.85 BAP(+) PLK3 180.82 BAP(+) PTK2 (FAK) 4 0.90 BAP(+) PTK2B (FAK2) 2 0.84 BAP(+) PTK6(Brk) −3 0.82 BAP(+)* RAF1 (cRAF) −3 0.81 Y340D Y341D BAP(+) RET 12 0.77BAP(+) ROCK1 2 0.86 BAP(+) ROS1 5 0.83 BAP(+) RPS6KB1 0 0.78 (p70S6K)BAP(+) SGK (SGK1) 21 0.90 BAP(+) SYK 14 0.84 BAP(+) TBK1 −5 0.78 BAP(+)TEK (Tie2) 26 0.80 BAP(+) TYRO3 (RSE) 3 0.92 BAP(+) YES1 18 0.78 BAP(+)CDK7/cyclin −17 0.82 H/MNAT1 BAP(+) CDK9/cyclin T1 26 0.92 BAP(+) GSG2(Haspin) 36 0.83 BAP(+) PIK3CG (p110 38 0.87 gamma) BAP(+) SPHK1 83 0.62F1 + F2 + F3 + F4 AURKA (Aurora 62 0.86 A) F1 + F2 + F3 + F4 AURKB(Aurora B) 66 0.70 F1 + F2 + F3 + F4 FRAP1 (mTOR) 91 0.85 F1 + F2 + F3 +F4* MAP3K8 (COT) 64 0.73 F1 + F2 + F3 + F4 GSG2 (Haspin) 32 0.85 F1 +F2 + F3 + F4 PIK3CG (p110 48 0.58 gamma) F1 + F2 + F3 + F4** SPHK1 1000.75 F5 AURKA (Aurora 26 0.69 A) F5 AURKB (Aurora B) 20 0.70 F5 FRAP1(mTOR) 7 0.85 F5* MAP3K8 (COT) 38 0.73 F5 GSG2 (Haspin) −2 0.85 F5PIK3CG (p110 1 0.80 gamma) F5 SPHK1 33 0.75 F5-2 AURKA (Aurora 12 0.69A) F5-2 AURKB (Aurora B) 12 0.70 F5-2 FRAP1 (mTOR) −3 0.85 F5-2* MAP3K8(COT) 23 0.73 F5-2 GSG2 (Haspin) 3 0.85 F5-2 PIK3CG (p110 −16 0.80gamma) F5-2 SPHK1 15 0.82 F5C AURKA (Aurora 10 0.69 A) F5C AURKB (AuroraB) 7 0.70 F5C FRAP1 (mTOR) −7 0.85 F5C* MAP3K8 (COT) 3 0.73 F5C GSG2(Haspin) 4 0.85 F5C PIK3CG (p110 21 0.58 gamma) F5C SPHK1 −5 0.70 F53AURKA (Aurora 16 0.69 A) F53 AURKB (Aurora B) 14 0.70 F53 FRAP1 (mTOR)−2 0.85 F53* MAP3K8 (COT) 35 0.73 F53 ABL1 M351T 12 0.68 F53 ALK C1156Y−5 0.81 F53 EGFR (ErbB1) 8 0.83 d746-750 F53 KIT D816H 18 0.82 F53 KITN822K 14 0.89 F53 KIT Y823D 27 0.82 F53 TTK 65 0.93 F53 WEE1 6 0.71 F53GSG2 (Haspin) −9 0.85 F53 PIK3CG (p110 43 0.58 gamma) F53 SPHK1 −11 0.82GPC AURKA (Aurora 22 0.86 A) GPC AURKB (Aurora B) 13 0.70 GPC FRAP1(mTOR) −1 0.85 GPC* MAP3K8 (COT) 7 0.73 GPC GSG2 (Haspin) 14 0.85 GPCPIK3CG (p110 2 0.58 gamma) GPC SPHK1 −5 0.70 F5-2-1 AURKA (Aurora 410.69 A) F5-2-1 AURKB (Aurora B) 34 0.70 F5-2-1 FRAP1 (mTOR) 5 0.85F5-2-1* MAP3K8 (COT) 64 0.73 F5-2-1 GSG2 (Haspin) −1 0.85 F5-2-1 PIK3CG(p110 0 0.80 gamma) F5-2-1 SPHK1 7 0.70 F5-2-2 AURKA (Aurora 25 0.69 A)F5-2-2 AURKB (Aurora B) 22 0.70 F5-2-2 FRAP1 (mTOR) −2 0.85 F5-2-2*MAP3K8 (COT) 40 0.73 F5-2-2 GSG2 (Haspin) −8 0.85 F5-2-2 PIK3CG (p110 110.58 gamma) F5-2-2 SPHK1 20 0.70 F5-2-3 AURKA (Aurora 6 0.71 A) F5-2-3AURKB (Aurora B) 15 0.70 F5-2-3 FRAP1 (mTOR) 16 0.85 F5-2-3* MAP3K8(COT) 44 0.73 F5-2-3 GSG2 (Haspin) 8 0.85 F5-2-3 PIK3CG (p110 12 0.58gamma) F5-2-3 SPHK1 −16 0.70 F5-2-5 AURKA (Aurora 10 0.71 A) F5-2-5AURKB (Aurora B) 15 0.70 F5-2-5 FRAP1 (mTOR) 2 0.85 F5-2-5* MAP3K8 (COT)11 0.73 F5-2-5 GSG2 (Haspin) 5 0.86 F5-2-5 PIK3CG (p110 12 0.58 gamma)F5-2-5 SPHK1 29 0.75 *ATP concentration of 100 μM **Test compound showedpossible interference with the acceptor

As can be seen, certain fractions of BAP may more effectively inhibit ofvarious kinases than compared to BAP(+) and BAP(−). For example, usingthe combination of fractions F1-F4 produced significantly greaterinhibition of the mTOR kinase than fraction F5 itself, or anysubstituents of F5. Moreover, BAP(+) shows significant inhibitoryactivity on various kinases over BAP(−). For example, BAP(+) showssignificant inhibitory behavior on SPHK1 kinase when BAP(−) or any otherfraction studied do not (the fraction F1+F2+F3+F4 interacted with theacceptor fluorophore in the experiment and prevented any emissionyielding a % inhibition of 100%).

Measurement of inhibition of various kinases was also performed bybinding a tracer to a kinase and the addition of Europium labeledanti-tag antibodies. When bound to the same kinase, the tracer andEuropium interact through FRET and fluoresce. Binding of the tracer andantibody to a kinase results in a high degree of FRET, whereasdisplacement of the tracer with a kinase inhibitor results in a loss ofFRET. This method of measurement removes the cleavage step necessary inthe above FRET measurement.

The kinase inhibitory activity of various 10 μM solutions of variousfractions was measured (LANTHASCREEN Eu Kinase Binding Assay ProfilingService available from Life Technologies). In these measurements,greater percent displacement indicates greater kinase inhibition. Table14 shows the percent displacement values and corresponding Z primevalues of several fractions, and of BAP(+) and BAP(−). The average %displacement is representative of two independent measurements.

TABLE 13 Average % Fraction Name Kinase Tested Displacement Z′ BAP(−)ABL1 H396P 11 0.70 BAP(−) ABL1 M351T 23 0.79 BAP(−) ABL1 Q252H 32 0.69BAP(−) ALK C1156Y 11 0.90 BAP(−) ALK F1174L 9 0.86 BAP(−) ALK L1196M 100.75 BAP(−) ALK R1275Q 9 0.91 BAP(−) EGFR 3 0.60 (ErbB1) d746-750 BAP(−)EPHA3 4 0.91 BAP(−) FGFR1 7 0.91 V561M BAP(−) FGFR3 15 0.84 G697C BAP(−)FLT3 ITD 5 0.64 BAP(−) KIT A829P 5 0.74 BAP(−) KIT D816H 28 0.72 BAP(−)KIT D816V 16 0.86 BAP(−) KIT D820E 9 0.96 BAP(−) KIT N822K 15 0.87BAP(−) KIT Y823D 12 0.74 BAP(−) MAP3K14 8 0.85 (NIK) BAP(−) MET D1228H 20.84 BAP(−) RET G691S 0 0.59 BAP(−) RET M918T 2 0.54 BAP(−) RET V804M 80.66 BAP(−) STK33 −2 0.90 BAP(−) TNK2 (ACK) 15 0.96 BAP(−) TTK 39 0.82BAP(−) WEE1 11 0.73 BAP(+) ABL1 H396P 19 0.70 BAP(+) ABL1 M351T 37 0.61BAP(+) ABL1 Q252H 30 0.69 BAP(+) ALK C1156Y 36 0.90 BAP(+) ALK F1174L 230.86 BAP(+) ALK L1196M 18 0.75 BAP(+) ALK R1275Q 15 0.91 BAP(+) EGFR 560.60 (ErbB1) d746-750 BAP(+) EPHA3 1 0.91 BAP(+) FGFR1 13 0.91 V561MBAP(+) FGFR3 31 0.77 G697C BAP(+) FLT3 ITD 13 0.79 BAP(+) KIT A829P 100.74 BAP(+) KIT D816H 44 0.59 BAP(+) KIT D816V 21 0.86 BAP(+) KIT D820E19 0.96 BAP(+) KIT N822K 33 0.89 BAP(+) KIT Y823D 45 0.74 BAP(+) MAP3K1426 0.85 (NIK) BAP(+) MET D1228H 2 0.84 BAP(+) RET G691S 8 0.59 BAP(+)RET M918T 73 0.54 BAP(+) RET V804M −13 0.66 BAP(+) STK33 7 0.90 BAP(+)TNK2 (ACK) 8 0.96 BAP(+) TTK 97 0.82 BAP(+) WEE1 34 0.73 F1 + F2 + F3 +F4 ABL1 M351T 31 0.68 F1 + F2 + F3 + F4 ALK C1156Y 42 0.81 F1 + F2 +F3 + F4 EGFR 97 0.86 (ErbB1) d746-750 F1 + F2 + F3 + F4 KIT D816H 370.82 F1 + F2 + F3 + F4 KIT N822K 57 0.89 F1 + F2 + F3 + F4 KIT Y823D 1080.82 F1 + F2 + F3 + F4 TTK 105 0.93 F1 + F2 + F3 + F4 WEE1 64 0.71 F5ABL1 M351T 31 0.60 F5 ALK C1156Y 11 0.81 F5 EGFR 11 0.83 (ErbB1)d746-750 F5 KIT D816H 22 0.82 F5 KIT N822K 9 0.89 F5 KIT Y823D 26 0.82F5 TTK 74 0.93 F5 WEE1 8 0.71 F5-2 ABL1 M351T −2 0.68 F5-2 ALK C1156Y 60.81 F5-2 EGFR 2 0.83 (ErbB1) d746-750 F5-2 KIT D816H 8 0.82 F5-2 KITN822K 2 0.89 F5-2 KIT Y823D 6 0.82 F5-2 TTK 36 0.93 F5-2 WEE1 16 0.71F5C ABL1 M351T 3 0.68 F5C ALK C1156Y −3 0.81 F5C EGFR 2 0.83 (ErbB1)d746-750 F5C KIT D816H 8 0.82 F5C KIT N822K 0 0.89 F5C KIT Y823D 4 0.82F5C TTK 30 0.93 F5C WEE1 1 0.71 F5-3 ABL1 M351T 12 0.68 F5-3 ALK C1156Y−5 0.81 F5-3 EGFR 8 0.83 (ErbB1) d746-750 F5-3 KIT D816H 18 0.82 F5-3KIT N822K 14 0.89 F5-3 KIT Y823D 27 0.82 F5-3 TTK 65 0.93 F5-3 WEE1 60.71 GPC ABL1 M351T 4 0.68 GPC ALK C1156Y 4 0.81 GPC EGFR −1 0.83(ErbB1) d746-750 GPC KIT D816H 1 0.82 GPC KIT N822K −1 0.89 GPC KITY823D 2 0.82 GPC TTK 24 0.93 GPC WEE1 5 0.71 F5-2-1 ABL1 M351T 32 0.68F5-2-1 ALK C1156Y 32 0.81 F5-2-1 EGFR 17 0.83 (ErbB1) d746-750 F5-2-1KIT D816H 52 0.82 F5-2-1 KIT N822K 22 0.89 F5-2-1 KIT Y823D 50 0.82F5-2-1 TTK 70 0.93 F5-2-1 WEE1 29 0.70 F5-2-2 ABL1 M351T 9 0.68 F5-2-2ALK C1156Y 14 0.81 F5-2-2 EGFR 7 0.83 (ErbB1) d746-750 F5-2-2 KIT D816H22 0.82 F5-2-2 KIT N822K 9 0.89 F5-2-2 KIT Y823D 25 0.82 F5-2-2 TTK 470.93 F5-2-2 WEE1 11 0.71 F5-2-3 ABL1 M351T −9 0.60 F5-2-3 ALK C1156Y 160.81 F5-2-3 EGFR −7 0.93 (ErbB1) d746-750 F5-2-3 KIT D816H 13 0.82F5-2-3 KIT N822K −9 0.89 F5-2-3 KIT Y823D 22 0.82 F5-2-3 TTK 76 0.93F5-2-3 WEE1 5 0.70 F5-2-5 ABL1 M351T 9 0.68 F5-2-5 ALK C1156Y 8 0.81F5-2-5 EGFR 4 0.93 (ErbB1) d746-750 F5-2-5 KIT D816H 7 0.82 F5-2-5 KITN822K −2 0.89 F5-2-5 KIT Y823D 5 0.82 F5-2-5 TTK 21 0.93 F5-2-5 WEE1 −10.71

As can be seen, certain fractions of BAP(+) provide inhibition ofvarious kinases including important targets for cancer therapeutics suchas TTK.

As can be seen foregoing, the present invention provides forcompositions effective in pharmaceutical compositions used in thetreatment of various cancers. The examples described herein areindicative of the beneficial effects possible with the describedpharmaceutical compositions. However, it should be understood that suchdescription is solely for convenience and clarity and is not intended tobe limiting in scope.

1. A pharmaceutical composition comprising an effective amount of aplurality of bioactive phospholipids derived from hen egg yolk and oneor more pharmaceutically acceptable diluents, excipients and/orcarriers, wherein said plurality of bioactive phospholipids issubstantially free of N-acyl ether phosphatidylethanolamines (NAEPE). 2.The pharmaceutical composition according to claim 1, wherein saidplurality of bioactive phospholipids is substantially free ofphosphatidylethanolamines (PE).
 3. The pharmaceutical composition ofclaim 1, wherein said plurality of bioactive phospholipids issubstantially free of lysophosphatidylcholines (LPC).
 4. Thepharmaceutical composition according to claim 1, wherein said pluralityof bioactive phospholipids is substantially free ofglycerophosphocholine.
 5. The pharmaceutical composition according toclaim 1 wherein said plurality of phospholipids is enriched in one ormore lipids selected from the group consisting of: (i) one or morephosphatidylcholine (PC) derivatives having the structure:

where R₁ or R₂ are independently selected from the group consisting ofpalmitoyl (C16:0), hexadecenoyl (C16:1), palmitoleoyl (C16:1), stearoyl(C18:0), octadecenoyl (C18:1), oleoyl (C18:1), octadecadienoyl (C18:2),linoleoyl (C18:2), α-linolenoyl (C18:3), octadecatrienoyl (C18:3),arachidonoyl (C20:4), eicosatetraenoyl (C20:4), docosapentaenoyl(C22:5), and docosahexaenoyl (C22:6) radicals; and/or (ii) one or moresphingomyelin (SPH) derivatives having the structure

wherein R₆ is palmitoyl (C16:0), stearoyl (C18:0), oleoyl (C18:1),tetradecanoyl (C14:0), heptadecanoyl (C17:0), nonadecanoyl (C19:0),eicosanoyl (C20:0), docosanoyl (C22:0), docosenoyl (C22:1), henicosanoyl(C23:0), tetracosadienoic (C24:2), or tetracosanoyl (C14:0); andpharmaceutically acceptable salts thereof.
 6. The pharmaceuticalcomposition according to claim 5, wherein R₁ is selected from, palmitoyl(C16:0), palmitoleoyl (C16:1), stearoyl (C18:0), oleoyl (C18:1), andlinoleoyl (C18:2).
 7. The pharmaceutical composition according to claim5, wherein R₂ is selected from linoleoyl (C18:2), oleoyl (C18:1),eicosatetraenoyl (C20:4), and docosahexaenoyl (C22:6).
 8. Thepharmaceutical composition according to claim 5, wherein R₆ is palmitoyl(C16:0).
 9. The pharmaceutical composition according to claim 5, whereinsaid one or more phosphatidylcholine (PC) derivatives collectivelycomprise at least about 70% (w/w) of said plurality of phospholipids.10. The pharmaceutical composition according to claim 5, wherein saidone or more sphingomyelin (SPH) derivatives collectively comprisebetween about 0.1% and 25% (w/w) of said plurality of phospholipids. 11.The pharmaceutical composition according to claim 5, wherein saidplurality of phospholipids consists of one or more phosphatidylcholine(PC) derivatives and one or more sphingomyelin (SPH) derivatives;wherein said one or more phosphatidylcholine (PC) derivativescollectively comprise at least about 70% (w/w) of said plurality ofphospholipids and said one or more sphingomyelin (SPH) derivativescollectively comprise between about 0.1% and 25% (w/w) of said pluralityof phospholipids.
 12. The pharmaceutical composition according to claim5, wherein said plurality of phospholipids is enriched in one or morephosphatidylcholine (PC) compounds and one or more sphingomyelin (SPH)compounds.
 13. The pharmaceutical composition according to claim 5,wherein said plurality of phospholipids is enriched in a)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate and b)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate.
 14. The pharmaceuticalcomposition according to claim 13, wherein said plurality ofphospholipids is enriched in(2S,3R,E)-2-heptadecanamido-3-hydroxyoctadec-4-en-1-yl(2-(trimethylammonio)ethyl) phosphate.
 15. The pharmaceuticalcomposition according to claim 5, wherein said plurality ofphospholipids is enriched in a)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate and b)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, c)(R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, d)(R)-2((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, and e)(2S,3R,E)-2-heptadecanamido-3-hydroxyoctadec-4-en-1-yl(2-(trimethylammonio)ethyl) phosphate.
 16. The pharmaceuticalcomposition according to claim 5, wherein said plurality ofphospholipids is enriched in a)(R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, b)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate, c)(R)-2((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate, d)(R)-2,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate, e)(R)-2-(((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, f)(R)-2((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, g)(R)-2((7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, h)(R)-2((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, i)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, j)(R)-2-((Z)-octadeca-9-enoyl)oxy)-3-((Z)-hexadec-9-enoyl)oxy)propyl(2-(trimethylammonio)ethyl) phosphate, k)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(oleoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, l)(R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, m)(R)-2-(((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, n)(R)-2-(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate, and o) (2S,3R,E)-2-heptadecanamido-3-hydroxyoctadec-4-en-1-yl(2-(trimethylammonio)ethyl) phosphate.
 17. The pharmaceuticalcomposition according to claim 16, wherein said plurality ofphospholipids is further enriched in p)(R)-2-(oleoyloxy)-3-(palmitoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate, q) (R)-2,3-bis(oleoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate, and r) (R)-2-(oleoyloxy)-3-(stearoyloxy)propyl(2-(trimethylammonio)ethyl) phosphate.
 18. The pharmaceuticalcomposition according to claim 1, wherein said effective amount is fromabout 1 mg to about 2000 mg.
 19. The pharmaceutical compositionaccording to claim 1, wherein said effective amount is from about 10 mgto about 1200 mg.
 20. The pharmaceutical composition according to claim1, wherein said effective amount is from about 100 mg to about 1000 mg.21. The pharmaceutical composition according to claim 1, wherein saidhen egg is of the genus Gallus.
 22. The pharmaceutical compositionaccording to claim 1, wherein said hen egg is from the species Gallusgallus domesticus.